Phytochemicals as Potential Chemopreventive and Chemotherapeutic Agents for Emerging Human Papillomavirus–Driven Head and Neck Cancer: Current Evidence and Future Prospects

Head and neck cancer (HNC) usually arises from squamous cells of the upper aerodigestive tract that line the mucosal surface in the head and neck region. In India, HNC is common in males, and it is the sixth most common cancer globally. Conventionally, HNC attributes to the use of alcohol or chewing tobacco. Over the past four decades, portions of human papillomavirus (HPV)-positive HNC are increasing at an alarming rate. Identification based on the etiological factors and molecular signatures demonstrates that these neoplastic lesions belong to a distinct category that differs in pathological characteristics and therapeutic response. Slow development in HNC therapeutics has resulted in a low 5-year survival rate in the last two decades. Interestingly, HPV-positive HNC has shown better outcomes following conservative treatments and immunotherapies. This raises demand to have a pre-therapy assessment of HPV status to decide the treatment strategy. Moreover, there is no HPV-specific treatment for HPV-positive HNC patients. Accumulating evidence suggests that phytochemicals are promising leads against HNC and show potential as adjuvants to chemoradiotherapy in HNC. However, only a few of these phytochemicals target HPV. The aim of the present article was to collate data on various leading phytochemicals that have shown promising results in the prevention and treatment of HNC in general and HPV-driven HNC. The review explores the possibility of using these leads against HPV-positive tumors as some of the signaling pathways are common. The review also addresses various challenges in the field that prevent their use in clinical settings.


INTRODUCTION
Head and neck cancer (HNC) constitutes a large group of cancers arising in different anatomical sites of the head and neck (HN) region, comprising the lip and oral cavity, larynx, nasopharynx, hypopharynx, oropharynx, nasal cavity, paranasal sinuses, and salivary glands. Over 90% of these neoplastic tissues are squamous cell carcinomas (SCCs). According to WHO estimates for 2019, HNC was one of the leading forms of cancer with 931,931 new cases, representing 4.9% of all cancer cases (Globocan, 2020). Lip and oral cavity cancer made up nearly 40% of the total HNC cases followed by the cancer of the larynx region. Mortality statistics reported by GLOBOCAN estimate 467,125 deaths due to head and neck cancers, representing 4.7% of all cancer deaths. Prevalence data for 2020 point to India as carrying the highest burden of head and neck cancer, with 143,242 cases, followed by China (100,871), the United States of America (51,533), and the Russian Federation (23,772). These numbers are alarming and draw attention to immediate action against this highly preventable cancer as the etiological agents are well known.
Tobacco use, excessive alcohol consumption, and lately, infection of human papillomavirus (HPV) are the established risk factors for HNC (Marur and Forastiere, 2016). The risk of HNC is 10-fold higher in smokers than that of HNC in nonsmokers (IARC, 2004). Although excessive alcohol consumption is an independent risk factor, it also increases the risk for smokers (Smith et al., 2004;Chaturvedi et al., 2015). In the past decade, however, there has been a shift in the anatomic distribution of HNC with an increasing occurrence of neoplastic lesions in the oropharynx (Sturgis and Cinciripini, 2007). A concordant decrease in smoking prevalence and increase in HPV prevalence has been noted, especially in the younger agegroup. The review of clinical manifestations of HNC based on their anatomical, histological, and etiological factors revealed a dichotomy in treatment response (Aggarwal et al., 2020). The data strongly point toward existence of two distinct types of HNC, namely, one that is caused by tobacco and alcohol abuse or occupational exposure to various carcinogens, and the other which is caused by biological agents like infection of HPV and possibly the EBV. The evidence presented in the present manuscript suggests discrete differences among the two disease groups, with each requiring separate clinical management.
Most patients with HNC seek clinical intervention at advanced stages of the disease (Haddad and Shin, 2008). This trend is quite common in individuals of low socioeconomic status, who cannot afford expensive medical/surgical treatments. Despite a wellstandardized treatment regimen, current therapy has a very low success rate as 30-60% of patients diagnosed develop recurrent locoregional cancer or second primary cancers even after complete remission (Hashim et al., 2019). A major underlying factor is onset of chemo/radioresistance and treatment failure (Nikolaou et al., 2018). Thus, better therapeutic options are needed to mitigate this challenge. Moreover, prevention of HNC at an early precancer/cancer stage could be another window of opportunity by which disease burden and mortality due to HNC could be reduced.
Currently, prevention focuses on risk behavior reduction like cessation of tobacco and early diagnosis of the disease. However, there is an unmet need for new therapeutics that could effectively eliminate HNC cells, reduce the onset of chemo/radioresistance, and could prevent the progression of the disease.
Recently, there has been a renewed interest in phytochemicals and herbal derivatives with therapeutic correlates from traditional medicine in the treatment and prevention of HNC due to their safety, availability, efficacy, and low cost. A number of studies carried out to investigate screening of phytochemicals using different HNC cell lines, animal models, and clinical evaluation in patients showed potent anticancer activities in a small set of phytochemicals. However, very limited number of studies addressed the impact of these herbal derivatives on HPV infection and HPV-positive HNC. In this article, we have systematically reviewed the existing data on various phytochemicals demonstrating chemotherapeutic and chemopreventive activities against HNC with a special emphasis on phytochemicals/herbal derivatives that showed anticancer effects against HPV-positive HNC. Further, major deficiencies and actionable leads in this field have been highlighted.

HEAD AND NECK CANCER SPECTRUM
HNC is a group of neoplastic diseases that can be broadly classified based on their anatomical site, histological origin, and etiological factors ( Figure 1A). Figure 1B illustrates the anatomic sites of the HN region. Broadly, the HN area is classified into four regions, namely, the oral cavity, pharynx, nasal cavity, and larynx. The oral cavity consists of the vestibule (the area between the teeth and mucosa of the lips and cheeks) and the oral cavity proper. The oral cavity proper is the interior region of the mouth: the region between the two dental arches and majorly occupied by the tongue (Akintoye and Mupparapu, 2020). Soft palate and hard palate separates the oral cavity from the nasal cavity.

Anatomical Site-Specific Classification
Oral SCC (OSCC) arises from mucosal areas of the lips, front 2/3rd of the tongue, gums, internal lining of cheeks and lips, floor of mouth below the tongue, hard palate, and the area behind the wisdom teeth (Gartner, 1994), and constitutes a major proportion of cancers of the HN region. Globally, lip and oral cavity cancer prevalence is 34.7% among the overall cases of HNC. Lip and oral cavity cancer has the highest incidence in South-Central Asia (Globocan, 2020). The prevalence of lip and oral cavity cancer in the past 5 years is the highest in India, with a total burden of 300,413 cases. In the oral cavity proper, the tongue accounts for 40% of intraoral carcinomas (Neville and Day, 2002).
The pharynx is a channel located in the region of the neck midline. The pharynx is majorly classified into three regions: the nasopharynx (located posterior to nasal cavity), oropharynx (posterior end of oral cavity), and hypopharynx (behind the opening of larynx) (Albahout and Lopez, 2021). Globally, among HNC, the prevalence of the nasopharynx is 15.8%, which is the highest among three regions, followed by oropharynx 10.7%, which is higher than hypopharynx 5.5%. The incidence rate of the nasopharynx is high in Southeastern Asia, whereas the incidence rate of the hypopharynx is high in Central and Eastern Europe (Globocan, 2020). Incidence rates for the oropharynx are high in Europe, which is linked with alcohol consumption, tobacco smoking, and HPV. Incidence of HPV infection in the oropharyngeal region is rising at an alarming rate (Wierzbicka et al., 2021).
The nasal cavity is the upper most part of the respiratory tract. The nasal cavity is surrounded by four types of paranasal sinuses: frontal sinuses, sphenoid sinuses, paired maxillary sinuses, and ethmoid sinuses. Paranasal sinus malignancies are rare, accounting for less than 3-5% of the total HNC (Patel, 2017). The nasal cavity and paranasal sinuses disease burden are not covered by (Globocan, 2020) under HNC.
The internal space of the larynx is a pyramid shaped about 5 cm long, connecting the pharynx to the trachea and is a part of the respiratory system. According to Globocan (2020), the incidence of larynx cancer is highest in Central and Eastern Europe. Laryngeal cancer constitutes around 21.4% among HNC (Globocan, 2020).
Exocrine glands and salivary glands function to secrete saliva in the oral cavity. Three type of salivary glands are present: parotid gland (situated front of both ears), submandibular gland (posterior of the mandible), and sublingual gland (floor of the oral cavity) (Ghannam and Singh, 2021). In the salivary gland, majority tumors are benign, whereas malignant tumors are generally mucoepidermoid carcinoma and adenocarcinoma. Primary SCC is rare and aggressive in salivary glands, specifically in the parotid gland (Flynn et al., 1999). The incidence of the salivary gland cancer has been reported to be the highest in Middle Africa. Salivary gland cancer constitutes 6.6% of total HNC.

Histological Classification
In the oral cavity, the mucosa is of masticatory, specialized, and mobile type. It covers around 25% of the oral cavity. In order to understand mechanical forces caused by mastication, it is covered by specialized, orthokeratinized, stratified squamous epithelium. Depending on the anatomic site, over 60% of the mucosa in the oral cavity is lined by the stratified squamous epithelium. The upper surface of the tongue is lined by specialized mucosa, with numerous lingual papillae (Winning and Townsend, 2000).
Histologically, the tumors of the HN region are classified as carcinoma, adenocarcinoma, lymphoma, and blastoma depending upon the tissue from where they are originating (Ologe et al., 2005). For instance, cancer originating in squamous cells in the HN region is collectively termed as HNSCC, and the one originating in salivary glands is of glandular origin and classified as an adenocarcinoma. The most common cancer affecting the HN region is epithelial carcinoma, which constitutes 80-90% of total cases, followed by lymphomas and blastomas accounting for the rest (Ologe et al., 2005;Gilyoma et al., 2015). Among carcinomas, squamous cell carcinoma constitutes 67.7% of total carcinoma cases, whereas other carcinomas like follicular carcinoma, adenocarcinoma, adenoid cystic carcinoma, clear cell carcinoma, mucoepidermoid carcinoma, and malignant melanoma cover the remaining carcinoma cases (Adeyemi et al., 2008).
Carcinomas mostly spread in the regions of the larynx, nasopharynx, and least in maxillofacial bones and oral cavity regions, whereas predominant anatomical sites for lymphomas were lymph nodes, followed by the maxillofacial bones. In contrast, sarcomas occurred most frequently in the maxillofacial bones, face/scalp, and the nose area (Adisa et al., 2011). The distribution of these tumors varies among the agegroup of the patients. Most of the carcinomas are detected in the age-group of 45-64 years in contrast to sarcomas frequently occurring in the age-group of 25-44 years (Adeyemi et al., 2008;Adisa et al., 2011).
In the oral cavity, leukoplakia (white plaque) and its variants, erythroplakia (fiery red patch) and submucous fibrosis (most prevalent in India), are three conditions that are highly associated with the development of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Malignant transformation rates of leukoplakia range from 8.9 to 17.5 percent (Silverman et al., 1984;Lind, 1987). The buccal mucosa had the highest incidence of leukoplakia, with 18% of lesions, but had the lowest rate of malignant transformation (3%). The tongue accounted for 16% of lesions but had the highest rate of transformation at 24% (Warnakulasuriya and Ariyawardana, 2016). Erythroplakia occurs mainly in the middle aged and the elderly and has the prevalence ranging from 0.02 to 1%. Soft palate, floor of the mouth, and buccal mucosa have their highest rate of incidence. The reason for etiopathogenesis has not been determined, but chewing tobacco and consuming alcohol have been implicated as factors for the development erythroplakia. The malignant transformation rate in erythroplakia is very high (14-50%) (Reichart and Philipsen, 2005). Oral submucous fibrosis is another chronic and potentially malignant disorder characterized by juxtaepithelial fibrosis of the oral cavity. This lesion has been reported to have a malignant transformation rate of 7-30%. Its incidence is highly associated with the chewing of betel quid containing areca nut (Ranganathan et al., 2004).
Dysplasia can be categorized as mild (architectural disturbance and cytological atypia in lower third of the epithelium), moderate (architectural disturbance and cytological atypia in middle third of the epithelium), and severe (architectural disturbance and cytological atypia in greater than two-third of the epithelium). This classification of dysplasia by the WHO is referred to as the gold standard for histological diagnosis of oral potentially malignant disorders (OPMDs). The WHO defines OPMDs as "clinical presentation that carry a risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or in clinically normal mucosa" (Muller, 2018). Epithelial dysplasia, an important precursor of malignant transformation in the HN region, can be defined as a change in morphological characteristics of the epithelium, including architectural and cytotoxic changes and loss of differentiation of keratinocytes toward the surface. It involves replacement of a part or the entire epithelium by cells showing cellular atypia (Tilakaratne et al., 2019;Wils et al., 2020).
The stratified squamous epithelium lines the pharynx to protect it from mechanical stress. The pharynx and larynx both are lined with the ciliated pseudostratified columnar epithelium with goblet cells. A study suggests that lesions such as erythroplakia at high-risk sites in the oropharynx should be considered as invasive carcinoma or carcinoma in situ at high-risk sites unless a biopsy proves otherwise (Mashberg and Samit, 1995). However, the vocal cords are lined with the stratified squamous epithelium (Stiblar-Martincic, 1997). Although there is no consensus, premalignant lesions of the larynx are usually classified as chronic laryngitis, erythroplakia, leukoplakia, and erythroleukoplakia (Gale et al., 2009). In the premalignant and malignant lesions of the larynx, severe dysplasia and carcinoma in situ occur at the rate of 10-20% (Hellquist et al., 1982). The nasal mucous membrane is lined with the sensory epithelium with olfactory cells and the respiratory epithelium. The mucosa is rich in mucus-producing goblet cell. Nasal drainage is facilitated by the ciliated epithelium. Premalignant lesions of paranasal sinuses differ from other lesions of the HN region and are present as inverted papillomas. This cancer goes undiagnosed before the onset of symptoms. Malignant tumors of paranasal sinus are diagnosed at stages T3-T4 in two-thirds of cases. Additionally, in paranasal cancer, 10% of total SCCs and 4% of all adenocarcinomas have some degree of cervical lymph node involvement (Jegoux et al., 2013). Salivary glands constitute three cell types, namely, acinar cells, myoepithelial cells, and ductal cells (Brazen and Dyer, 2020). In the parotid gland, 70% of the tumors detected are benign. In the submandibular gland, adenoid cystic carcinoma is the common malignancy (16%). Sublingual gland tumors are rare but have the highest frequency of malignancy, ranging from 70 to 90% (Carlson and Schlieve, 2019).

Classification Based on Etiological Type
Tobacco-associated HNC: Association of tobacco and alcohol use with the onset of HNC is well established (IARC, 2004). Tobacco use is the leading cause of preventable death in the world. Tobacco smoking alone is the leading cause of cancer and cancer-related deaths worldwide. Nearly 85% of HNC are linked with tobacco use. Within the HN region, it has been conclusively shown to directly cause oral cavity, laryngeal, and pharyngeal cancer (Centers for Disease Control and Prevention, 2004). The International Agency for Research on Cancer (IARC) has classified carcinogens in groups, group 1: tobacco smoking, secondhand smoking, and smokeless tobacco for HNCs, which are sufficient for evident carcinogenicity in human (IARC, 2004). In developed countries, most inhaled or "mainstream" tobacco smoke comes from the use of manufactured cigarettes. Cigarettes burn at very high temperature and produce smoke that includes toxins and carcinogens. Similar drawbacks are with cigars, pipes, and water pipes (IARC, 2004).
Tobacco smoke contains a variety of group 1 carcinogens, namely, arsenic and benzene, but research is more focused on tobacco-specific N-nitrosamines, especially N-nitrosonornicotine and 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone, as they are established carcinogens. In HNC of HNSCC type, the latter one is more associated with increasing the risk of cancer development (Oreggia et al., 1991). Tar is another compound which is linked with an increased risk of HNC (Franceschi et al., 1992).
Studies have shown that development of HNC is strongly related with dose-dependent tobacco smoking but can also occur with low daily usage (Berthiller et al., 2016). Moreover, the duration of exposure also significantly affects the risk of HNC. The risk of daily smoking for more than 30 years was found to be more carcinogenic (Cohen et al., 2018).
Alcohol-associated HNC: HNC is also associated with alcohol abuse. Studies suggest that alcohol consumption and cigarette smoking are differentially associated with the risk of HNSCC subtypes (Bagnardi et al., 2001). A large prospective study has confirmed that alcohol consumption is strongly linked to HNSCC (Freedman et al., 2007). Among all, oropharyngeal SCC (OPSCC) is the most associated, while laryngeal SCC (LSCC) is the least associated with heavy alcohol consumption (Zeka et al., 2003;Lubin et al., 2009;Toporcov et al., 2015). Clinically, there is no distinction between alcohol-and tobacco-associated HNC.
Occupational exposure-associated HNC: Apart from smoking of tobacco products, occupational exposure to dusts from wood, textiles, leather industries, flour, nickel, chromium, fumes from rubbing alcohol (also called isopropyl alcohol), radium, glue, formaldehyde as well as solvent fumes used in furniture and shoe production, and asbestos are the main risk factors for sinonasal carcinomas. Hypopharygeal and laryngeal carcinoma are associated with the use of coal for heating or cooking (IARC, 2012). These tumors have an aggressive clinical behavior and resemble tobacco-associated tumors in progression and therapeutic response.
Epstein-Barr virus-associated HNC: The etiology and natural history of nasopharyngeal SCC (NPSCC) is closely linked to that of Epstein-Barr virus (EBV) infection. This neoplasm is an uncommon disease with very low prevalence in most countries (Wei and Sham, 2005). Although EBV infection is pervasive, NPSCC incidence differs considerably around the world (Chang and Adami, 2006). In most geographical regions where NPSCC is endemic, the onset of EBV infection occurs at an early age. The estimated latency period of this virus is around a decade, so other factors also contribute for NPSCC development. Evidences indicate that this cancer is predominant in individuals of Southeast Asian descent due to genetic differences (Chang and Adami, 2006;Bei et al., 2016;Liu et al., 2017).
HPV-associated HNC: HPV is a DNA virus with oncogenic potential associated with over a dozen genotypes referred to as high-risk HPV. Persistent HPV infection is chiefly associated with the development of anogenital and cervical carcinomas. HPV16 and HPV18 genotypes are the most prevalent carcinogenic types and act via action of two major oncogenes, E6 and E7. These oncogenes target cell cycle and promote tumor growth by targeting and downregulating p53 and pRb, respectively. Many molecular and epidemiological studies support association of HPV with HNC, especially with OPSCC (Franceschi et al., 1996). Over the last 125 years, observations  Syrjanen et al. (2017)).

Year
Milestones References

1891, 1896
First speculation of contagious nature of cutaneous warts Payne (1891), Jadassohn (1896) 1901 Contagious transmission of condyloma warts in the tongue after oral sex described Heidingsfield (1901Heidingsfield ( ) 1907 Viral etiology of oral lesions Ciuffo (1907Ciuffo ( ) 1923 Association of human wart virus with laryngeal warts established Ullman (1923Ullman ( ) 1943 Oral papillomatosis as a viral disease was established in rabbits Parsons and Kidd (1943) 1948, 1956 Reporting of koilocytotic atypia in laryngeal papilloma Ayre and Ayre (1949), Ishiji et al. (1992) 1978 Epithelial atypia in laryngeal papilloma reported Quick et al. (1978Quick et al. ( ) 1973 Identification of HPV in laryngeal papilloma Boyle et al. (1973Boyle et al. ( ) 1974 Detected virus-specific DNA in human tumors zur Hausen et al. (1975Hausen et al. ( ) 1976 HPV association with koilocytotic atypia established as a sign of HPV infection Meisels and Fortin (1976), Purola andSavia (1977) 1978 Development of noncommercial antiserum against HPVs Pyrhonen andNeuvonen (1978) 1980 HPV 6 was isolated from condyloma acuminata zur Hausen (1980) 1982 Expression of HPV structural proteins in laryngeal carcinoma Syrjanen et al. (1992Syrjanen et al. ( ) 1982 HP 11 was detected in laryngeal papilloma Gissmann et al. (1982Gissmann et al. ( ) 1982 HPV detection in benign and malignant oral SCC Jenson et al. (1982), Syrjanen et al. (1983aSyrjanen et al. ( ) 1983 An extensive squamous cell papilloma of the nasal cavity and also filling the entire left maxillary sinus is reported Syrjanen et al. (1983bSyrjanen et al. ( ) 1983 Morphologic and immunohistochemically features indicate HPV infection in OSCC Syrjanen et al. (1983aSyrjanen et al. ( ) 1987 HPV DNA in benign and malignant sinonasal lesions Syrjanen et al. (1987Syrjanen et al. ( ) 1989 Detection of HPV DNA in human oral tissue biopsies and cultures Maitland et al. (1989Maitland et al. ( ) 1989 HPV16 DNA detection in tonsillar carcinoma Brandsma andAbramson (1989) 1992 Success in preparation of virus-like particles (VLPs), namely, BPV1 and HPV16, that established HPV serology and vaccination Kirnbauer et al. (1992) 1992 First report showing the presence of transcriptionally active and integrated HPV infection with expression of E6/E7 mRNAs in tonsilar cancer Snijders et al. (1992) 1992 HPV16/18 DNA in nasopharyngeal carcinoma Dickens et al. (1992Dickens et al. ( ) 2004 Papillomaviruses recognized as a taxonomic family of their own De Villiers et al. (2004)  2005 Differential expression and activity of transcription factors in HPV-positive oral cancers Mishra et al. (2006Mishra et al. ( ) 2008 Halard zur Hausen was awarded with Nobel prize in physiology or medicine -Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 699044 speculating the presence of a virus transmitting oral tumors have matured and led to the identification of a subset of HNC with distinct clinical presentation that show an early onset (Table 1). Approximately 35% of all HNC and 77% of tonsillar cancers harbor HPV, with greater than 60% of cases being the HPV16 subtype (McKaig et al., 1998). A significant variation in HPV prevalence in HNC types is recorded within different studies and from different geographical regions (Gillison et al., 2015). Finding HPV in the HN region is paradoxical. However, a sexual mode of transmission has been suggested. Due to mucoepithelial tropicity of these viruses, if the virus gets access to these tissues via opportunistic contact with infected genital organs, it can result in the establishment of HPV infection in the HN region ( Figure 2). Patients with other HPV-associated neoplasms or premalignant conditions are presumed to be at a higher risk of HNC development. Among spouses, women having a history of cervical dysplasia showed higher incidence of HPV-related oropharyngeal cancer (Hemminki et al., 2000). Patients with a history of anogenital cancer have shown a higher risk of tonsillar cancer (Frisch and Biggar, 1999). These HPV-positive cancers are primarily SCCs in their histological manifestations.

Recognition of Human Papillomavirus-Positive HNSCC as a Distinct Human Papillomavirus-Driven Subtype
Data emerged in last 2 decades strongly support the recognition of HPV-positive HNSCC as a distinct disease with a well-defined clinical and molecular pattern and unique risk factors ( Table 2). These HPV-positive tumors were reported in early stage (Pintos et al., 1999;Smith et al., 2004;Hammarstedt et al., 2006), well differentiated histology (Pintos et al., 1999;Gupta et al., 2015), basaloid morphology (Gillison et al., 2000), larger tumors (Gletsou et al., 2018), and either no lymph node involvement (Pintos et al., 1999) or with cystic cervical lymph node positivity FIGURE 2 | Various routes and sites of HPV transmission. HPV is a sexually transmitted infection that can be received through horizontal transmission (heterosexual intercourse, anogenital, and oro-genital) and in some rare cases through various fomites, and inoculation via fingers, clothes, utensils, towels, and surgical instruments. (A) Vertical transmission (perinatal transmission: mother to baby) and oral-oral transmission of HPV could be the main source of nonsexual transmission of HPV in oral sites. (C) Most prevalent high-risk and low-risk HPV types reported in benign and malignant lesions of the HN region are depicted with representative figures where the relative frequency of the each type is indicated with corresponding sizes. (B) [Disclaimer: Pictures used to make composite diagram and to represent HPV types are derived from different internet sources and does not claim to be the original representation of the indicated HPV type] (Sarkola et al., 2008;Chaturvedi et al., 2015;Fu et al., 2015;Visalli et al., 2016;Louvanto et al., 2017;Sabeena et al., 2017;Syrjanen, 2018;Houlihan et al., 2019).
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 699044 6   Poorly differentiated carcinomas (72.2%) Higher expression and DNA binding activity of AP-1 and NF-κB with c-fos and Fra-2; and p50 and c-rel as the major binding partners forming the functional AP-1 and NF-κB complex, and selective participation of p65 Low expression and DNA binding activity of AP-1 with c-Jun as the major binding partners forming the functional AP-1 complex Induced expression of p65 and p27 leading to well differentiation and better prognosis Participation of c-Rel with p50 that in crosstalk with AP-1/Fra-2 leading to poor differentiation and aggressive tumorigenesis Gaykalova et al. (2015) Tissues from HNSCC patients (n 195) and noncancer-affected patients (n 63) [discovery-HPV (16) (Goldenberg et al., 2008). These tumors showed low risk of second primary malignant neoplasm (Adjei Boakye et al., 2018) with a better overall and disease-free survival (Ragin and Taioli, 2007;Fakhry et al., 2008;Ang et al., 2010;Rischin et al., 2010;Posner et al., 2011;Fakhry et al., 2014). Irrespective of the tissue subtype involved, HPV positivity in HNSCC emerged as a strong biomarker associated with better prognosis (Gillison et al., 2000;Wookey et al., 2019). HNSCC is overrepresented in males (Pintos et al., 1999;Gillison et al., 2008). The gender bias increases further in HPV-positive tumors (Pintos et al., 1999). Gender-specific data derived from HPV-positive oropharyngeal cancer (OPC) patients showed a higher risk of premalignant lesions in men (Ryerson et al., 2008). These observations are indicative of a tumor-promoting role of either male-specific hormones leading to differences in clearance of HPV infections due to the endocrine-immune interactions (Klein, 2000), or a distinct cellular environment in oral mucosal cells of men that promote transcriptional activation of viral oncogenes and HPV-mediated HNC. An increased anal HPV16/18 prevalence has been noticed, which correlated with high free testosterone levels in men having sex with men (Hsu et al., 2015).
Time trend studies carried out in different cohorts and registries particularly in North America and Europe revealed an interesting disease dynamics among all the HN sites (Chaturvedi et al., 2008;Chaturvedi et al., 2011). OPC showed a characteristic change in incidence (Hammarstedt et al., 2006;Mehanna et al., 2013). During the 30 year period, HPV-negative OPC declined steeply with a simultaneous and more prominent emergence of HPV-positive OPC (Chaturvedi et al., 2011). HPVnegative OPC and non-OPC that included all other HN sites are HPV-unrelated and traditionally linked to smoking and alcohol abuse. On the contrary, the studies showed a definitive and strong link of HPV-positive tumors with the oral-genital sexual contact . The HPV-positive HNSCC shows large variations in prevalence among different geographical regions (De Martel et al., 2019) and may be associated with prevailing sociocultural and sexual practices, whereas genetic predispositions that may also play a sizable role in this phenomenon cannot be ruled out. In line with these observations, a higher incidence of HPV-positive tumors in Hispanic population has been reported .
Early studies repeatedly pointed to a lower median age of HPV-positive HNSCC (Chaturvedi et al., 2008;Posner et al., 2011). However, a recent study demonstrated increased HPV positivity even in older age-group (Windon et al., 2018), thus indicating that early onset of HNSCC was merely circumstantial. Reviewing the factors contributing to the changing pattern of HNSCC over last 50 years revealed a major shift in societal practices with respect to depiction of sexuality (Syrjanen et al.,   . Surprisingly, in 1969, Denmark legitimized display of explicit content, which was followed by the Netherlands and Sweden, and by 1972, the United States observed a peak in the films displaying oral sexual acts. Therefore, the shift in the HNSCC from HPV-negative to HPV-positive tumors observed in the western population is possibly associated with two independent phenomena that occurred simultaneously. First, establishment of tobacco's carcinogenic potential (Vizcaino et al., 2015) and consequent implementation of anti-tobacco policies; and second, display of oral sex on motion pictures that promoted indulgence in high-risk behavior leading to increased exposure of oral mucosa to genital HPV infections. Treatment efficacy can be maintained by evaluating the HPV-positivity in OPSCC; as they have better prognosis, they can be treated with less aggressive treatment to avoid serious side effects to reduce treatment-associated toxicities in relatively younger patients (Boscolo-Rizzo et al., 2016).

Molecular Signatures of Human Papillomavirus-Positive Head and Neck Cancer
During first 2 decades, research was emphasized on the detection of HPV and its distribution in the HN region. Subsequent studies revealed a series of distinctive molecular features in HPV-positive HNC ( Table 3). In HPV-positive tumors, wild-type p53 was functionally active and was downregulated by E6 oncoprotein. Reduced p53 transcript was associated with the activation of many oncongenic pathway genes, which contributes to genetic instability in the development of cancer (van Houten et al., 2001;Licitra et al., 2006). HPV-positive HNC lesions show characteristically high expression of p16 INK4a , which serves as a surrogate marker for HPV (Licitra et al., 2006). In contrast, HPV-negative tumors showed inactivating p53 and p16 INK4a mutation in HNSCC.
Based on transcription milieu, HNC showed constitutively active nuclear factor-κB (NF-κB) irrespective of their HPV status. However, a detailed molecular dissection of the constitutively active NF-κB complex showed the presence of p50:p65 heterodimer in HPV-positive tumors, whereas homodimer of p50:p50 was found in HPV-negative tumors (Mishra et al., 2006;Gupta et al., 2018). Similarly, in HNC tumors for constitutively active AP-1, JunB and JunD were involved with c-Fos and Fra-2 in HPV-positive HNC, whereas in HPV-negative HNC, c-Jun was the major binding partner .
STAT3, another transcription factor that is linked with carcinogenic outcome, was strongly associated with HPVnegative HNC and was characteristically low in HPV-positive tumors (Gaykalova et al., 2015;Verma et al., 2017). SOX2 amplification was observed in HPV-negative HNC, while there was no amplification in HPV-positive HNC (Schrock et al., 2014). HPV-positive HNCs were immunologically more active with high infiltration of T and B lymphocytes and myeloid dendritic cells, and had higher M1-type macrophages along with high chemokine production and PD1 expression (Partlova et al., 2015;Chen et al., 2018). A detailed discussion of various differentially expressed carcinogenically relevent genes in HPV-positive and HPV-negative HNC that contribute to better prognosis was described earlier (Aggarwal et al., 2020).

Current Treatment Strategies Against Head and Neck Cancer
Treatment of HNC requires a multi-modality approach depending on the stage and site of the tumor (Marur and Forastiere, 2008). Early tumors are treated with surgery or radiation, whereas intermediate-and late-stage tumors benefit from a combined modality approach. Due to essential requirement of clear margins in surgery, it is an option only for early tumors; still it carries a risk of cosmetic deformity and impaired function (Kofler et al., 2014). A study on the quality of life after oropharyngeal surgery reports high incidence of fatigue, reduced sexuality, difficulty in swallowing and other teeth, salivary gland, and mouth-opening-related problems (Bozec et al., 2018). Surgery also requires additional treatment to reduce the risk of locoregional and distant failure in advancedstage HNC (Porceddu et al., 2004). Platinum-based chemotherapy has been central in treating HNC. Combinatorial therapies with or without platinum drugs have been proven superior in terms of the response rate and the ability to tackle drug resistance than platinum-based chemotherapy treatment. Targeted therapies using monoclonal antibodies such as cetuximab, against epidermal growth factor receptor (EGFR) either in combination with a standard chemotherapy regimen or as a single agent, have also proven effective to some extent to treat HNC. But these approaches also bear side effects apart from the development of chemoresistance in a short period of time (Price and Cohen, 2012). These therapies have a myriad of debilitating toxic effects such as nephrotoxicity, hepatotoxicity, and cardiotoxicity. Also, various cardiac events have been reported, like arrhythmias, myocarditis, and cardiomyopathy, to congestive heart failure (Hartmann and Lipp, 2003).
Radiation therapy (RT) is often performed as an adjunct to surgery or in concurrence with chemotherapy (Marur and Forastiere, 2016). Wendt et al. reported a 3-year overall survival rate of 24% in RT arm vs. 48% in RT plus CT arm in stage III/IV HNC, whereas the 3-year locoregional control rate was 17% in RT arm and 36% in RT plus CT arm (Wendt et al., 1998). However, a long-term toxicity risk to the salivary glands, pharyngeal constrictor muscles, and thyroid gland, leading to xerostomia, dysphagia, percutaneous endoscopic gastrostomy tube dependence, chronic aspiration, and hypothyroidism, had been observed (Langendijk et al., 2008).
Despite a clear prognostic advantage and better response to therapy, therapeutic management for HPV-positive HNC is almost the same as that of any HPV-negative HNC. Considering the younger age of the patients, there have been efforts to reduce the long-term toxicity of anticancer treatment without risking the survival benefits (Kofler et al., 2014). Reduction in dose of radiotherapy, use of cetuximab (Marur et al., 2017) instead of cisplatin for chemoradiation, and transoral robotic surgery (TORS) are a few efforts to mention that are specifically directed to HPVpositive HNC. Considering HPV-positive tumors to be immunologically active, in recent past, attempts have been made to design PD1-PDL1 immunotherapeutic strategies (Qiao et al., 2020). New cancer immune-prevention treatments include FDA-approved inhibitory antibodies such as pembrolizumab (anti-PD1 mAb), nivolumab (anti-PD1), and ipilimumab (anti-CTLA-4 mAb) (Bauman and Ferris, 2014;Ferris et al., 2018;Mehra et al., 2018;Havel et al., 2019); co-stimulation and co-inhibition pathways (Kuss et al., 2003;Tsukishiro et al., 2003;Baruah et al., 2012;Pardoll, 2012); and check-point blockade therapy (Davis et al., 2016;Muzaffar et al., 2021). A systematic assessment of the cost effectiveness of ICIs showed nivolumab was not cost-effective over chemotherapy for HNC (Verma et al., 2018). Moreover, none of these approaches target HPV. A study attempted to develop Trojan vaccine against HPV could not show significant benefit of therapeutic vaccines against HPV in HNC (Voskens et al., 2012). A recent study showed a chimeric HPV16 E7 DNA vaccine induced prophylactic and therapeutic efficacy in a cervical cancer mouse model, but its effect on HPVpositive HNC remains to be examined (Garza-Morales et al., 2019).

Therapeutic Challenges in Head and Neck Cancer Management
Despite aggressive treatment and organ preservation with current clinically administered curative therapies, the overall 5-year survival is less than 50% (Forastiere et al., 2013). With existing heterogeneity in the origin, poor response rates and substantial systemic toxicity associated with current standard-of-care treatment of advanced HNC remain a significant challenge (De Lartigue, 2015). As molecular targeted therapies come into clinical use, the great interindividual variability in the efficacy of these compounds highlights the absolute need to determine predictive factors of tumor and toxic responses to these new therapeutic agents (Bozec et al., 2009). Further, patients with locally advanced or recurrent HNC present a separate therapeutic challenge. Treatment options are limited, and morbidity can be substantial. Surgical intervention has debilitating effect on normal daily routine and patient psychology. Recurrent HNC is difficult to treat for multiple reasons, including the effects of prior treatment on tumor cells and normal tissues, as well as the infiltrative and multifocal nature that typically characterizes recurrent disease in this area (Ho et al., 2014).
Limitations of these therapies have prompted clinical and translational research for better chemotherapeutics with less treatment-associated toxicities. Many studies are focusing on biologically active compounds from herbal origin to develop chemotherapeutic agents with fewer side effects and higher efficacy (Seo et al., 2015;Kunnumakkara et al., 2017). Many of these phytochemicals can serve as alternatives for chemotherapy sensitizers (Bharti and Aggarwal, 2017;Huang and Yu, 2017).

Emerging Chemotherapeutic Phytochemicals/Herbal Derivatives Against Head and Neck Cancer
Phytochemicals have found relevance in HNC therapy because natural compounds provide a cost-effective, safe, and less toxic alternative to synthetic drugs currently in wide use. Effectiveness of various phytochemicals as therapeutic agents has been well documented in the literature, and they are now widely being studied as potential agents to treat and prevent HNC. Many preclinical studies have successfully demonstrated the anticancer activity of pure and well-characterized phytochemicals and herbal derivatives on cells obtained from different HN regions using in vitro and in vivo experimental systems (Table 4). However, a majority of these studies employed cell lines derived from the oral cavity, so the data may be slightly skewed.

Moscatilin
In vitro: FaDu (Hypopharyngeal SCC) Cell cytotoxicity: carcinogenic role in HNC (Song and Grandis, 2000). Guggulsterone, a biosafe nutraceutical, phosphorylated p65 and inhibited tobacco smoke and nicotine-induced NF-κB and pSTAT3 proteins and their downstream targets COX-2 and VEGF (Macha et al., 2011). Dihydroartemisinin is a known phytochemical, which is effective as an antimalarial agent, induces DNA double-strand break and promoted oxidative stress, and decreases pSTAT3 nuclear localization which successively increases autophagic cell death (Shi et al., 2017).
In 90% HNC, the PI3K/AKT/mTOR pathway is upregulated (Marquard and Jucker, 2020). Whenever ligand-like growth factors bind with RTKs, they dimerize and lead to the activation of intercellular tyrosine kinase. PI3K partially activates Akt through PIP3 and PIP2. Then to stimulate full activity of Akt, mTORC2 phosphorylates its carboxy-terminal. Akt functions by phosphorylation that leads to the activation or suppression of many proteins involved in cell proliferation, growth, and cell motility (Brazil and Hemmings, 2001;Chaisuparat et al., 2016). Wogonin, a flavonoid compound, has anticancer activity which induces autophagy by LC3 I/II cleavage and inhibits mTOR/P70S6K and Raf/ERK, which in turn inactivates PI3K/Akt and induces apoptosis in NPC cells (Chow et al., 2012). Urosolic acid downregulated Akt/mTOR signaling and expression of NF-κB, which further downregulates ERK and MMP-2 in OSCC cells .
Loss of carcinogenic signaling was associated with reduced cell survival mechanisms. Honokiol, a phytochemical from Magnolia plant, reduced the level of Bcl-xL protein, while Bax expression in xenograft HNC tumors increased. It also reduces the expression of mTOR and its downstream p70S6K (Singh et al., 2015). Similarly, (-)-gossypol, a polyphenol, was reported to bind to Bcl-xL that inhibited HNC proliferation (Wolter et al., 2006).
Antiproliferative activity of phytochemicals was associated with various degrees of cell cycle arrest in most of these studies. Cell cycle-regulating molecules such as cyclins and cdks were downregulated by oridonin, chrysophanol, lupeol, honokiol, and proanthocyadins. Piperine, a nitrogenous pungent substance, induced cell cycle arrest in the G2/M phase and induced apoptosis by changing mitochondrial membrane potential and by activating caspase-3 (Siddiqui et al., 2017). Chrysophanol, a secondary metabolite, downregulated the expression of cyclinD1, CDK4, cdc2, and CDK2, and arrested cell cycle at the G1 phase. It also induced cell death by ROS production (Hsu et al., 2020). Similarly, lupeol induced cell cycle arrest in the G1 phase by increasing the expression of p53, Bax, and CDKN2A, and downregulating cyclin D1 (Bhattacharyya et al., 2017). Oridonin, a bioactive diterpenoid, induced apoptosis by regulating Bax/Bcl-2 and activating caspases. It also decreased cell proliferation by downregulating PI3K/Akt/mTOR pathways. By regulating cyclins, it arrested cells in the G2/M phase (Yang et al., 2018b). Even though the end effect was antiproliferative, the mechanism of action of these phytochemicals differed significantly.
In in vivo studies, the phytochemicals were tested in murine models, where nude mice were implanted with OSCC cell lines. These mice were used to measure the effect of phytochemical on tumor growth. Tumors from euthanized mice were examined for their size and volume. ECGC (Yoshimura et al., 2019), gossypol (Wolter et al., 2006), quercentin (Zhang et al., 2019), proanthocyadins (Prasad and Katiyar, 2012), tanshinomes (Qiu et al., 2018), shikonin , β elemene (Huang and Yu, 2017), and bitter melon extract (Rajamoorthi et al., 2013) depicted reduction in size and volume of tumor xenografts, and inhibition of xenograft growth. Inhibition of growth was also observed in the ex vivo study with lupeol.
Some of the phytochemicals were also tested in clinical trials; however, these studies are very limited ( Table 5) and emphasize an urgent unmet need in this area to harness the translational potential of emerging phytochemicals. Lippman et al., 1988 conducted a phase II randomized study with 13-cis-retinoic acid (isotretinoin) (3 mg/kg/day) and methotrexate (15 mg/m 2 on the first three days in a 3-week cycle) among 40 patients with advanced SCCs. They achieved a response rate of 16% with isotretinoin, which included a complete response, a partial response, and a minor response. In the methotrexate-treated group, however, the response rate was 5%. The median survival rate from the start of treatment was also lower in the methotrexate group (4 months) than that in the isotretinoin group (4.5 months) (Lippman et al., 1988). Another phase I study with isotretinoin by Weisman et al., 1998 reported its strong synergetic relationship with cisplatin. The maximum tolerated dosage as determined by the study (20 mg/day) was able to attain a complete response at the primary site in all of the 10 evaluable patients (Weisman et al., 1998). There are very few clinical trials on therapeutic potential of phytochemicals in HNC because of lack of interest from pharma industry due to low cost of the molecules, and clinical trial requires a lot of investments. Also, HNC patients with advance stage tumor do not participate in therapeutic clinical trials as it may risk the available therapeutic benefits of existing therapies; however, use of phytochemicals as adjunct therapies may prove beneficial in long run as they will not compromise the benefits of participating patients. Nevertheless, more in vivo studies are needed to screen promising leads into clinical trials.

Emerging Chemopreventive Phytochemicals/Herbal Derivatives Against Head and Neck Cancer
Cancer chemoprevention refers to the use of agents to retard the progression of carcinogenesis, reverse, or inhibit it. The aim of chemoprevention is to lower the risk of developing invasive or clinically significant disease. Chemopreventive phytochemicals thus seek to occasion a chemopreventive response when the primary tumors have not reached a critical size, or seek to block and reverse development of a diagnosed premalignant tumor, or prevent metastasis and growth of metastatic tumors (Tosetti et al., 2002). Angiogenesis, which refers to the biological process of vessel formation, also plays a crucial role in cancer progression. Angiogenesis is also responsible for transition of a dormant tumor to a malignant state (Sogno et al., 2009). An early intervention could possibly prevent cancer formation by regulating "angiogenic switch," the point at which the tumor induces angiogenesis. Thus, angiogenesis is a critical target for chemoprevention (Tosetti et al., 2002).
A battery of phytochemicals reportedly possess cell invasion, migration, angiogenesis, and metastasis inhibitory activities ( Table 6). These phytochemicals exhibit these antitumor activities by regulating the expression of various molecules such as metalloproteinases (MMPs), especially MMP-2 and MMP-9, which affect cancer migration and invasion. Some MMPs also exhibit proangiogenic properties as they can activate proangiogenic factors such as VEGF, and angiopoietin (Folgueras et al., 2004). These phytochemicals were also observed to regulate the MAPK/ERK pathway, which plays a crucial role in cell proliferation .
In vivo studies with nobiletin on male BALB/c nude mice suppressed tumor formation and metastasis by downregulating NF-κB translocation, MMP-2, and TIMP-2 proteins, and decreased phosphorylation of ERK1/2 (Chien et al., 2015). Toona sinensis crude extract decreased the incidences of SCCs, tumor number, tumor volume, and tumor burden in male Syrian golden hamsters by downregulating protein levels of survivin, XIAP, PCNA, iNOS, and COX-2 . Delayed tumor initiation incidence was reported in bitter melon extract-fed mice (Sur et al., 2018). Oral lesion incidence decreased in 4NQO exposed mice after being fed a black raspberry diet by downregulating PKA-AMPK pathway genes, which regulates mitochondrial functions (Knobloch et al., 2019).
Most of the reported clinical trials focusing on chemoprevention in HNC have been conducted on oral premalignant lesions (Table 7). Historically, clinical studies conducted on HNC chemoprevention with natural agents have centered on the use of retinoid. Bichler et al. (1983) reported that serum levels of retinol, RBP, and PACB were significantly lower in patients with carcinomas of the HN region (Bichler et al., 1983). This was considered to be of significance in tumor development studies, and since then, it has been corroborated by various research groups such as by Kapil et al. (2003). One of the initial studies conducted with retinoids was by Hong et al., 1986. The double-blind study demonstrated the effectiveness of 13-cRA in reducing the size of oral premalignant lesions in 44 patients. In a study conducted by Stich et al. (1988a) on 65 patients having well-developed oral leukoplakia, a complete remission in the lesions was observed in 57.1% of patients in the vitamin A group as compared to 3% of patients in the placebo group (Stich et al., 1988a). An interesting study was also conducted by Mathew et al., (1995) using lyophilized Spirulina fusiformis, an effective source of dietary vitamin A and other micronutrients (Mathew et al., 1995). A 1 g/day dose of oral Spirulina fusiformis powder demonstrated an effective chemoprevention activity by producing a complete response in 20/44 subjects in the treatment group as compared to 3/43 subjects in the placebo group. A partial response was observed in five patients in the Spirulina fusiformis treatment group as compared to zero in the placebo group.
A study by Stich et al. (1988b) reported on the combined effect of beta-carotene and vitamin A on betel quid chewers in India with well-established leukoplakias. Remission in the group receiving combined treatment was 27.5% as compared to 14.8 and 3% in groups receiving just beta-carotene and the placebo, respectively. The rate of new leukoplakia occurrence was also found to be higher in the beta-carotene (14.8%) and placebo groups (21.2%) that that of new leukoplakia occurrence in the group treated with both betacarotene and vitamin A (7.8%) (Stich et al., 1988a). The effectiveness of beta-carotene as a chemopreventive agent was also established by Garewal et al., (1990), who in study with 25 patients achieved a response rate of 71% in the group treated with 30 mg/day betacarotene (Garewal et al., 1990). A comparative study conducted in two phases with beta-carotene and isotretinoin by Lippman et al. (1993) reported that low-dose isotretinoin therapy was significantly more active against leukoplakia than beta carotene when preceded by high-dose induction therapy . In another three-arm double-blind study conducted with 160 patients by Sankaranarayana et al. (1997), the vitamin A and beta-carotene arms attained a complete regression of leukoplakia lesions in 52 and 32% of the subjects, respectively, as compared to just 10% in the placebo arm (Sankaranarayanan et al., 1997).
Two clinical studies conducted with retinyl palmitate by Jyothimayi et al. (1996) and Issing et al. (1996) reported a complete inhibition of the formation of secondary primary tumors (SPTs) and a complete remission of leukoplakia lesions in 75% of participants in drug-receiving arms, respectively (Issing et al., 1996;Jyothirmayi et al., 1996). Significant decrease in the prevalence odds ratio of oral leukoplakia was observed by Zaridze et al. (1993) in a double-blind trial conducted among 532 subjects with various combinations of riboflavin, retinol, vitamin E, and beta-carotene (Zaridze et al., 1993). Another combinatorial study with betacarotene, ascorbic acid, and alpha-tocopherol by Kaugars et al. (1994) noted a clinical improvement in 55.7% of the participants; 48.8% of people who continued their pre-study levels of risk factor exposure showed improvement (Kaugars et al., 1994).        • Cell migration and invasiveness ↓ • ERK and p38 phosphorylation ↓ • Anti-metastatic activity

Gallic acid
In A 24-week study by Benner et al. (1993) using alpha-tocopherol as a single agent to treat patients with oral leukoplakia attained a clinical response in 20 patients from the 43 patients who had signed up for it (Benner et al., 1993). Alpha-tocopherol was part of yet another study by Shin et al. (2001), when delivered with IFN-α and 13-cis-retinoic acid; among 44 patients evaluable at a median 24-month follow-up, 9% had locoregional recurrence, 5% had both locoregional recurrence and distant metastases, and 2% developed an SPT. The overall survival rate at the 24-month follow-up was noted to be 91% (Shin et al., 2001).
Green tea, a widely consumed beverage, has been previously reported to exhibit chemopreventive properties against cancer (Imai et al., 1997). Since it inhibits tumor development, is nontoxic, and is easily available to the general population, it has been a subject of interest in cancer studies. Two clinical studies where green tea was used as an agent to treat precancerous lesions like leukoplakia were included. Li et al. (1999) reported a decrease in lesions in 37.9% patients in the tea-receiving arm as compared to improvement in lesions of only 10% patients in the placebo arm (Li et al., 1999). Tsao et al. (2009) reported a dose-dependent clinical response by randomizing 41 patients in three green tea extract-receiving arms (dosage: 500 mg/m 2 , 750 mg/m 2 , 1,000 mg/m 2 ) and 1 placebo arm, with a clinical response in 50% of patients in the three combined arms and a 58% clinical response rate in the two combined higher dose arms (Tsao et al., 2009). They also reported a histological improvement in lesions after treatment.
Lycopene is a carotenoid that is abundant in a human diet and has been associated with a reduced risk of cancer of the upper digestive tract (De Stefani et al., 2000). Singh et al. (2004) reported a dose-dependent response of oral leukoplakia for administration of lycopene, with clinical improvement observed in 80% of patients receiving 8 mg/day lycopene; 66.3% patients receiving 4 mg/day dose showed a clinical response . A clinical study with lycopene and Calendula officinalis by Singh and Bagewadi (2017) reported a reduction in the average size of lesions posttreatment. The mean difference in the reduction in size before and after treatment for Group I was 2% ± 1.0 cm, while for the Group II, it was 1.6% ± 0.9 cm (Singh and Bagewadi, 2017).
Curcumin, a flavonoid derived from Curcuma longa, has been extensively investigated for its pharmacological properties. It is known to have antioxidant, anti-inflammatory, and anticancer properties, and thus is a promising phytochemical for HN region chemoprevention. A randomized double-blind phase IIB study by Kuriakose et al. (2016) on 223 patients with oral leukoplakia reported a clinical response in 67.5% of patients in the curcumin arm (dosage: 3.6 g/day for 6 months) and a histological response in 22.5% of patients (Kuriakose et al., 2016). Sun et al. (2010) conducted a randomized placebo-controlled study with ZengShengPing; a mixture of six medicinal herbs was known to have pharmacological effects. 3.6 g of ZSP administered daily for 8-12 months was observed to produce a positive response in 67.8% of patients in the treatment arm as compared to 17% in the placebo group (Sun et al., 2010). Mallery et al. (2014) conducted a placebo-controlled clinical trial using topically applied 10% w/w black raspberry (BRB) gel among 40 patients with oral premalignant lesions. The study reported an average decrease of 26% in the size of BRB-treated lesions as compared to an increase in size by 18% in the placebo gel-applied lesions. Two patients in the BRB arm exhibited a complete lesional resolution as compared to zero in the placebo gel group (Mallery et al., 2014).
Although a large volume of data reflects targeting of key procarcinogenic signaling pathways by various phytochemicals, none of them directly address their possible impact on HPV infection or in HPV-positive HNC lesions. Therefore, we specifically looked for evidences where phytochemicals have been tested against HNC cells with HPV-positive background.

Chemotherapeutic and Chemopreventive Phytochemicals/Herbal Derivative With Anti-Cancer and/or Anti-Human Papillomavirus Activity in Head and Neck Cancer
Most of the studies described earlier lack specificity against HPV infection. The natural derivatives having both anti-HPV and anti-HNC activity hold great potential as chemotherapeutic and chemopreventive agents for HNC caused by HPV. However, there are only limited resources in terms of HPV-related HNC model systems. Unlike many other infections, HPV cannot be propagated in in vitro cultures or in animal models. Unfortunately, suitable animal models that mimic HPV-driven HNC do not exist. In such a scenario, HPV-positive HNSCC cell lines serve as a suitable in vitro system. There are currently only a limited set of HPV-driven HN cancer cell lines developed by different investigators ( Table 8). As of now, we could identify only 11 cell lines that have been described as HPV positive, and their HPV genotype has been confirmed. A majority of them have HPV16 positivity, and the genome was found to be integrated (Steenbergen et al., 1995;Ballo et al., 1999;White et al., 2007;Brenner et al., 2010;Ye et al., 2011;Tang et al., 2012;Kalu et al., 2017). Similarly, one cell line each of HPV18 and HPV33 has been reported (Owen et al., 2016;Kalu et al., 2017). Although there are various HNSCC cell lines described so far, their HPV status must be ascertained. These cells lines proved to be useful model systems as they showed p16 positivity and demonstrated higher radiosensitivity (Rieckmann et al., 2013). In these cell line integration of HPV from E1, E2, L1, L2, and LCR have been observed which recapitulate observation in primary tumors by whole genome sequence which suggests various hotspots for HPV integration events in HPV-positive tumors and that may play varied role in the development of HNC (Gao et al., 2019). These cell lines and tumor tissues showed the presence of the viral infection by the presence of viral DNA and transcripts which emerged as valuable tools (Steenbergen et al., 1995;Ballo et al., 1999;White et al., 2007;Brenner et al., 2010;Ye et al., 2011;Tang et al., 2012;Kalu et al., 2017).
A limited set of studies have been conducted to examine anti-HPV and anticancer activities in HNC ( Table 9). The evidence suggests that HPV-positive cells can serve as suitable tools for screening of anti-HPV and anti-HNC. Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), a green tea derivative, exhibits various chemopreventive effects, including inhibition • No significant toxicity attributable to beta-carotene was encountered in this trial Garewal et al. (1990) • completed study 24 • Adult patients diagnosed as having clinically measurable oral leukoplakia while undergoing routine dental examinations    Kaugars et al. (1994) • Gender bias M:F::45:34 • 20/44 subjects in the SF group showed a complete response as compared to 3/43 subjects in the placebo group Headache, muscular pain Mathew et al. (1995) • Completed study 87 SF:   of growth factor-mediated proliferation (Liang et al., 1997;Liang et al., 1999a), induction of G1 arrest (Khafif et al., 1998;Liang et al., 1999b;Liberto and Cobrinik, 2000), and apoptosis (Ahmad et al., 1997;Paschka et al., 1998;Yang et al., 1998;Li et al., 2000). In this study, it induced apoptosis via the mitochondrial pathway through decreasing the expression level of Bcl2 and Bcl-xL and simultaneously increasing the Bax expression level that in turn activates caspase-9 in HNC cell lines YCU-N861 and HPV18 transformant YCU-H891 cell line. Treatment with EGCG inhibited the phosphorylation of EGFR, STAT3, and ERK proteins. It also inhibited the basal and transforming growth factor α-stimulated c-fos and cyclin D1 promoter activity. It decreased the level of cyclin D1 and pRB, accounting for the cellular arrest in the G1 phase (Masuda et al., 2001). The efficacy of the therapies used for the treatment of HNC can be enhanced by the incorporation of EGCG in current therapeutic regimens. Currently, anti-EGFR antibodies or specific tyrosine kinase inhibitors are being used in combination with radiation and certain chemotherapy agents in clinical trials for various types of cancer, as inhibition of the EGFR-related signal transduction pathway enhances the cytotoxic effects of radiation or various chemotherapy agents (Wu et al., 1995;Dent et al., 1999;Bonner et al., 2000). Hence, EGCG may have certain advantages over EGFR antibodies or selected tyrosine kinase inhibitors, as it is relatively inexpensive, natural, and nontoxic, and hence might be useful in administering for a longer period without any adverse effects. Clinical efficacy of EGCG still needs to be determined, and the direct correlation between chemopreventive effect of EGCG and HPV activity is yet to be established by further in vitro and in vivo studies. Also, the p53 status during EGCG administration needs to be determined as 50% of HNC carry mutations in the p53 gene, which in turn can modulate effects of EGCG (Wang et al., 2020). Emodin, a natural trihydroxyanthraquinone, has lower oxidation-reduction potential than that of oxygen; hence, under hypoxic conditions, it can be reduced to cytotoxic agent, sensitizing the cells to irradiation (Zhu et al., 2005;Zou et al., 2010;Schwartz et al., 2011). It affected the NPC cell (CNE1, a HeLa contaminated cell line) promotion and progression by inducing oxidative damage by significantly increasing the expression level of ROS, which induces apoptosis and downregulates mRNA and protein levels of HIF-1α. It also reduces the promotion of survival of carcinoma cells and induces cell cycle arrest at the G2/M phase. Hence, exposure of NPC cells in vitro and xenografts in vivo to emodin enhanced their radiosensitivity (Hou et al., 2013). Therefore, incorporation of emodin, a bioreductive agent, represents a viable therapeutic strategy targeting HIF-1α, by enhancing cytotoxicity of chemotherapeutic drugs via modulation of redox status of cancer cells and multidrug resistance reversal (Yi et al., 2004;Brown et al., 2007;Cai et al., 2008;Huang et al., 2008). It may also serve as an effective radiosensor, thereby improving efficacy of radiation therapy in radiation-resistant cancer cells. Moreover, since emodin can effectively enhance the radiosensitivity in vivo, it holds a potential as a radiosensitizing drug for NPC patients in future. Still a direct correlation between emodin and HPVactivity needs to be established. Curcumin (diferulolylmethane), an active component of turmeric and a perennial herb, has been shown to suppress the expression of HPV oncogenes mediated by downregulation and reduced transactivation of AP-1 and NF-kB superfamily members, representing a novel mechanism regulating HPVinduced oral carcinogenesis (Li et al., 1993;Prusty and Das, 2005). Its effect was also observed in HPV-positive 93VU147T cells. The cell viability is reduced significantly. It also induces apoptosis by decreasing the expression level of antiapoptotic factors such as Bcl-2 and cIAP2, and inducing proapoptotic factors like Bax. It downregulated the protein expression of AP-1 members: c-Jun, JunD, and JunB along with NF-kB members, p50 and p65. Curcumin also tends to show HPV-specific effects like reducing the mRNA levels of HPV16/E6, which in turn inhibits E6-mediated p53 degradation. Hence, curcumin exhibits therapeutic potential for HPV16-mediated oral oncogenesis suppression (Mishra et al., 2015). Similar result was observed in a later study on curcumin as phytochemical having both anti-HNSCC and anti-HPV activities, which was carried out on UD-SCC-2, UPCI: SCC131, and UPCI:SCC84 cell lines. It affected cancer promotion, cellular proliferation, and progression. Curcumin inhibited cancer cell growth and orosphere formation ability. Also, it induced cytotoxic effect along with HPV-specific effects like decreasing the expression level of HPV16 E6/E7 oncoproteins, and downregulated mi-RNA21 expression  significantly in HPV-positive oral CSCs. Hence, curcumin can sensitize the HPV-positive oral CSCs, thus making the cancer treatment more effective when used in combination with standard anticancer drugs or radiation, depicting its potential as a therapeutic agent. Further studies are required for deciphering the therapeutic effects of curcumin by determining its solubility and bioavailability, mechanism(s) of action, and potential molecular targets (Bano et al., 2018). Sulforaphane, an isothiocyanate, derived from broccoli sprout extracts; treatment of HPV-negative HNC cell lines-UM-SCC-22A, UM-SSC-1, and CAL33-and HPV-positive cell line SSC090 led to dose-and time-dependent stimulation of NRF2 signaling for carcinogen detoxication. It also dephosphorylated inhibitedSTAT3 and promoted cell death. Similar effects were also observed in in vivo and clinical study including female C57BL/6 mice (5-6 weeks; 18 mice/group) and 10 human subjects, respectively. The pilot clinical trial demonstrated consistent bioavailability of sulforaphane, promising sustainable chronic administration. Although it is a costeffective and natural product, further studies planned with encapsulated broccoli extract are required to enhance the ease of acceptability and dispensing. Also, HPV-specific chemopreventive effects are yet to be determined (Bauman et al., 2016).
6-Gingerol, a β-hydroxy ketone, derived from ginger rhizome, inhibited tumor cell proliferation and induces cellular toxicity, cell cycle arrest, apoptosis, and caspase 3/7 activation, as observed in KB and SCC4 cells. Also, the caspase-3-dependent proapoptotic activity was stimulated. It also inhibited cell cycle progression arresting the cells in G2 and M phases. Hence, 6-gingerol can be considered as a safe and potent chemotherapeutic/chemopreventive compound acting via cell cycle arrest and induction of apoptosis (Kapoor et al., 2016). Further studies should be directed toward determination of the chemopreventive effects of 6-gingerol in in vivo conditions and clinical trials along with direct correlation with HPV activity.
Thymol, a monoterpene derivative phenol, is a TRPA1 agonist found in thyme and oregano. It inhibited cellular proliferation and exhibited long-lasting cytotoxic effects as observed in CAL27, SSC4, and SSC9 cell lines. It also inhibited tumor growth in vivo as observed in CAL27 and HeLa-derived mouse xenografts. It induces the activation of TRPA1 and apoptosis via the mitochondria-dependent pathway. It promoted mitochondrial dysfunction via reducing mitochondrial membrane potential significantly (De La Chapa et al., 2018). Its HPV-specific effects still need to be determined along with the determination of bioavailability and tolerability to understand its therapeutic effects for future incorporation into cancer treatment.
Apigenin, a flavonoid, found abundantly in flowers of plants, vegetables, and fruits, exerts anticarcinogenic effects via preventing malignant transformation of cells, regulating cell signal transduction pathways, increasing apoptosis, and modulating cell cycle (Fang et al., 2007;Zhao et al., 2011;Zhu et al., 2013;Salmani et al., 2017;Yang et al., 2018a). It inhibited cancerous cell migration ability and arrested them in the G1 phase as observed in SHEE cells induced by HPV-18 and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). It downregulated the expression of CDK4, cyclin D1, and pRB, affecting cell cycle. Apigenin also induced cellular apoptosis via caspase-3 activation and inhibits DNA alkylation. With low toxicity and various beneficial bioactivities, apigenin can be considered as a potential chemopreventive agent against cancers, particularly, in smokers with HR-HPV coinfection (Yin et al., 2020). Hence, most of the phytochemicals mentioned above showed anticancer activity in HPV-positive cells, where only a limited studies focused on HPV-specific effects. Thus, considerable attention should be paid to analyze the correlation between anti-HNC and anti-HPV activity of the phytochemicals as a chemopreventive and chemotherapeutic measure to prevent HPV-HNC.

Critical Issues Associated With the Use of Phytochemicals
Despite their encouraging pharmacological activities, there are bottlenecks in the translation of phytochemical-based therapies applicable in clinical settings.
Low bioavailability: Many phytochemicals suffer from having poor aqueous solubility and low retention in blood circulation. Pharmacological concentration of these phytochemicals in blood and tumor tissues is low because of poor absorption, high rate of metabolism, chemical degradation, and speedy clearance. It has been reported that serum levels of curcumin were quite low, reaching a maximum of 0.06 ± 0.01 μg/ml after oral administration of 500 mg/kg in rats . Ravindranath and Chandrasekhara (1980) also demonstrated that 40% of curcumin gets excreted unchanged in feces when orally administered to rats (Ravindranath and Chandrasekhara, 1980). A pilot study conducted among 10 healthy patients also reported poor bioavailability of sulforaphane with a regimen of topical exposure to sulforaphanerich broccoli sprout extracts (Bauman et al., 2016). Chen et al. (1997) investigated the plasma pharmacokinetics of EGCG in rats and found the oral bioavailability of only 1.6% after a 75 mg/kg oral dose and a 10 mg/kg intravenous dose . Similarly, circulation half-life of resveratrol when administered through i. v. was few minutes and showed rapid elimination (Marier et al., 2002), whereas EGCG and quercetin attain low concentrations in blood, which is inadequate for antitumor activity (Lagoa et al., 2017).
Obstacles associated with the use of phytochemicals for treating and preventing cancer can be overcome with advances in the field of nanotechnology. A 10-fold dose advantage was achieved without any loss of effectiveness by encapsulating ECGC in polylactic acid-polyethylene glycol nanoparticles (Siddiqui et al., 2009). Increased absorption was also reported by nanoparticle encapsulation of curcumin despite its low solubility in water. Additionally, curcumin loaded poly lacticco-glycolic acid nanoparticles increased the oral bioavailability to nine times that of the native form, with piperine as absorption enhancer (Shaikh et al., 2009). Further advancements in this field should be encouraged.
Toxicity: Although phytochemicals may show toxicity when administered in high doses, they exhibit less adverse effects than conventional therapies. In a clinical trial with 50 oral leucoplakia patients, significant toxicity, severe enough to cause withdrawal of 6 patients, was observed with the use of isoretinoin (Garewal et al., 1999). Additionally, not all phytochemicals are safe for consumption. It has been found that a few natural compounds such as capsaicin (chilli pepper), cycasin, and cycas seed are tumor-promoting and must be avoided (Bode and Dong, 2015). Moreover, unregulated use of phytochemicals may have a danger of contamination by potential carcinogens.
Pharmaceutical industry challenges: Pharma-research into phytochemicals and herbal derivatives has experienced a slow decline during the recent times (Koehn and Carter, 2005;Katiyar et al., 2012). This can be attributed to advancements in high-throughput screening technology against defined molecular targets, advances in genomics, molecular and cellular biology, development of combinatorial chemistry, and a declining importance among large pharma-companies on the commercial considerations of phytochemicals that are often associated with poor financial returns and nearly absent IPR protection. Unique features of natural compounds such as a greater number of chiral centers, higher number of oxygen atoms, and greater molecular rigidity pose further challenges for medicinal chemists as they develop analogs to reduce toxicity, improve absorption, or to improve the efficacy, which is often achieved by adding or deleting selected functional groups.
Poor independent agents: While phytochemicals may not be efficient as standalone chemotherapeutic agents, many groups have established their efficacy as adjuvants to traditional therapies. A study demonstrated the benefits of combining sulforaphane with cisplatin and 5-fluorouracil (Elkashty et al., 2018). Sulforaphane increased the cytotoxicity of cisplatin and 5fluorouracil by two-fold and ten-fold, respectively. It did not alter the viability and functions of noncancerous stem cells. Sulforaphane combined treatments successfully inhibited cancer stem cell colony formation, sphere formation, and tumor progression in vivo. In an Italian study conducted among 23 patients undergoing treatment with 5-fluorouracil and cisplatin, prolonged responses were reported with the use of retinol palmitate in chemotherapy intervals. Toxicity levels were acceptable, and treatment did not interfere with the quality of life (Recchia et al., 1993). A study also observed significant growth inhibition and enhanced apoptosis in HNC cells with the use of curcumin along with 5-fluorouracil or doxorubicin. The study thus demonstrated the significant potential of combining curcumin with 5-fluorouracil or doxorubicin as a treatment modality for HNC management (Sivanantham et al., 2016).
Preclinical efficacy vs. clinical response: The cause for discrepancy in effectiveness of phytochemical agents in preclinical and human clinical trials has been conjectured to arise because of differences in dosage, metabolic differences, bioavailability, differences in circulating tissue levels of chemopreventive agents in humans and animals, exposure conditions to damaged tissue vs normal tissue, follow-up time, and the assessed ends. Second, high doses are often administered to animals in contrast to low doses admisible to humans in clinical trials. Although animal models have significantly helped in the identification of carcinogens, and chemopreventive and chemotherapeutic agents, they are not available for every HNC organ site. Furthermore, existing models cannot mimic human exposure complexities of carcinogens, metabolic competence, turnover of cells, and their repair capacity.

CONCLUSION AND FUTURE PROSPECTIVE
Phytochemicals show immense potential in the field of HNC chemotherapy and chemoprevention agents. In this evolving landscape, the success of employability of phytochemicals depends on our ability to decipher their molecular mechanics. Using phytochemicals in combination with another or in conjunction with existing chemotherapeutic practices or an alternate therapy is an area worth exploring.
We have also observed that there has not been much phytochemical-related research on HPV-induced HNC. However, numerous phytochemicals that are effective against HPV-induced cervical cancer have been reported in the literature (Bharti et al., 2018). In today's era, therapies to distinguish HPVpositive HNC from HPV-negative HNC are required. As HPVpositive HNC has better outcomes, the tumors can be treated with well-established phytochemicals targeting the HPV-mediated carcinogenic mechanisms. Thus, it might be valuable to study whether these phytochemicals can find application in HNC treatment and prevention. Activity of these phytochemicals can be checked on HNC cell lines or in vivo in laboratory conditions and can also be screened by using bioinformatic tools. There is a strong requirement to develop HPV-based concurrent therapies so that HNC can be treated more effectively. There are many associated challenges with the use of natural compounds. In pharmacological doses, the adverse effects of these natural compounds such as increased toxicity and low bioavailability are amplified. For chemoprevention to be feasible in treating premalignant lesions, the compound must be well tolerated and have long-lasting benefit. Moreover, the various signaling pathways contributing to HNC tumorigenesis mandate the use of compounds with multiple molecular targets. It is noteworthy that molecular targets of many such phytochemicals in active HNC are now well known (Figure 4). It is also worthy to note that not many clinical studies have been conducted despite discovery of numerous phytochemicals with multiple molecular targets. In order to determine the safety and efficacy of phytochemicals, it is imperative that more of such clinical studies, with different phytochemicals, are funded and conducted. Challenges associated with the use of phytochemicals such as low bioavailibilty, and toxicity can be possibly overcome with the use of chemical analogs, adjuvant therapies, and nanoparticle delivery mechanisms. Hence, a number of studies on phytochemicals against HPV-driven HNC are now accumulating; a comparative account on their relative efficacy is needed and should be addressed to harness the potential of phytochemicals in clinical studies. Research in these areas needs encouragement for effective management of HPV-positive HNC in future.
RT radiation therapy RTK receptor tyrosine kinase S6K1 ribosomal protein S6 kinase beta-1 SCC squamous cell carcinoma Sin1 stress-activated protein kinase-interacting protein SLT smokeless tobacco SOX2 SRY (sex determining region Y)-box 2 STAT3 signal transducer and activator of transcription 3

TFs transcription factors
TIMPs tissue inhibitor of metalloproteinases