Progress of research on molecular targeted therapies for colorectal cancer

Colorectal cancer (CRC) is one of the most common malignancies, accounting for approximately 10% of global cancer incidence and mortality. Approximately 20% of patients with CRC present metastatic disease (mCRC) at the time of diagnosis. Moreover, up to 50% of patients with localized disease eventually metastasize. mCRC encompasses a complex cascade of reactions involving multiple factors and processes, leading to a diverse array of molecular mechanisms. Improved comprehension of the pathways underlying cancer cell development and proliferation, coupled with the accessibility of relevant targeted agents, has propelled advancements in CRC treatment, ultimately leading to enhanced survival rates. Mutations in various pathways and location of the primary tumor in CRC influences the efficacy of targeted agents. This review summarizes available targeted agents for different CRC pathways, with a focus on recent advances in anti-angiogenic and anti-epidermal growth factor receptor agents, BRAF mutations, and human epidermal growth factor receptor 2-associated targeted agents.


Introduction
Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related death worldwide. The onset of CRC is subtle and challenging to detect in early stages, underscoring the importance of timely screening. Moreover, approximately 22% of patients are diagnosed with metastatic disease at the outset. The 5-year metastatic CRC (mCRC) survival rate is approximately 15% (Howlader N et al., 2020). Therefore, precise treatment of mCRC using appropriate drugs based on relevant molecular biosignatures is essential to prolong the survival time of patients.
The main treatment strategies for mCRC are chemotherapy using cytotoxic agents, molecular-targeted therapy, and immunotherapy. Combination chemotherapy regimens frequently involve the use of fluorouracil (5-FU)-based drugs together with oxaliplatin or irinotecan. These combinations have demonstrated significant improvements in patient survival, with survival durations exceeding 20 months (Tournigand et al., 2004;Van Cutsem et al., 2014;García-Alfonso et al., 2021;Glimelius et al., 2021). Rapid and significant advancement in the development of targeted agents for CRC has been observed since the approval of cetuximab for mCRC treatment in 2004 ( Figure 1) Currently, more than 10 agents have been approved for mCRC treatment. Combined treatments utilizing targeted drugs and chemotherapy have been reported to increase the 5-year survival rate from 9% to 15% and survival to more than 30 months in mCRC patients (Van Cutsem et al., 2016;Siegel et al., 2022). As distant metastasis is the main cause of death in CRC, relevant review that comprehensively explores and summarizes the efficacy of molecularly targeted drugs in CRC and associated latest research progress does not exist, and this review highlights these. This review focuses on progress of research on targeted agents for treating mCRC patients, providing a reference for clinicians for precise treatment of CRC.
2 Classification and mechanism of common targets in CRC 2.1 Vascular endothelial growth factor (VEGF) target VEGF family includes VEGFA-D, VEGF receptor (VEGFR), and placental growth factor. VEGFA, commonly known as VEGF or vascular permeability factor, is the main angiogenic feature of the VEGF family. The role of VEGFB in tumor angiogenesis has not yet been elucidated; however, VEGFC and VEGFD mainly regulate lymphatic endothelial cell growth (Dvorak, 2002;Karkkainen et al., 2002;Ferrara et al., 2003;Bry et al., 2014). VEGFR includes VEGFR1-3, with VEGFA capable of binding to VEGFR1 and 2, and VEGFC and VEGFD binding to VEGFR2 and 3, respectively. VEGFR activation can promote cell proliferation, migration, and growth via the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K) pathways, leading to angiogenesis and tumor angiogenesis (Ivy et al., 2009). VEGF inhibitors used in CRC treatment include monoclonal antibodies (mAbs) like bevacizumab, ramucirumab, Ziv-aflibercept, as well as tyrosine kinase inhibitors (TKIs), such as sunitinib, sorafenib, fruquintinib, and regorafenib. These drugs target the VEGF pathway to inhibit angiogenesis and tumor growth (Figure 2).

EGFR and HER2 target
Epidermal growth factor receptor (EGFR), also known as ERBB1 or human epidermal growth factor receptor (HER) 1, is a member of the HER family, which also includes HER2-4. EGFR promotes tumor cell proliferation, differentiation, growth, and distant metastasis by activating downstream signaling pathways, such as PI3K and MAPK (Roskoski, 2014;Kumagai et al., 2021). EGFR activation induces the secretion of angiogenic factors, such as VEGF, which stimulate the formation of new blood vessels. Increased angiogenesis ensures sufficient blood supply to the tumor, facilitating its expansion and providing nutrients for sustained growth. Additionally, EGFR signaling provides anti-apoptotic signals through the activation of AKT, which inhibits apoptosis by inactivating pro-apoptotic proteins (De Luca et al., 2008). This allows cancer cells to evade programmed cell death and survive in unfavorable conditions. Furthermore, HER2 overexpression, as a member of the EGFR family, inhibits the tumor suppressor gene, p53 (Ménard et al., 2003). Common EGFR inhibitors used in cancer treatment include anti-EGFR mAbs, such as cetuximab and panitumumab, EGFR TKIs like gefitinib, erlotinib, and afatinib, as well as HER2 mAbs trastuzumab and pertuzumab, and HER2 TKI lapatinib. The inhibitors target EGFR and HER2, blocking their signaling pathways and inhibiting tumor growth and progression.

RAS/RAF/MEK/ERK target
The RAS/RAF/MEK/ERK signaling cascade (MAPK) pathway is one of the downstream EGFR pathways, which mainly regulates cell proliferation and differentiation. RAS family members include KRAS, NRAS, and HRAS (Karnoub and Weinberg, 2008). KRAS mutation (MT) accounts for approximately 32%-40% of CRC cases, and BRAF V600E MT accounts for 5%-15% (De Roock et al., 2011;Pakneshan et al., 2013). MEK, or MAPK/ERK kinase, plays a pivotal role as a mediator in the downstream signaling of the MAPK pathway. ERK, also known as extracellular signal-regulated kinase, is a critical component of this pathway (Downward, 2003). Studies have confirmed that MEK/ERK inhibitors significantly enhance the treatment efficacy in patients with KRAS/BRAF-mutant tumors (Hatzivassiliou et al., 2013;Morris et al., 2013). Available inhibitors targeting the RAS/RAF/MEK/ERK pathway include dabrafenib and vemurafenib as BRAF inhibitors, and trametinib and encorafenib as MEK inhibitors. Additionally, there are ERK inhibitors such as ulixertinib and temuterkib.

PI3K/AKT/mTOR target
PI3K is an enzyme that plays a crucial role in cellular signaling pathways involved in cell growth, survival, and metabolism. It functions by phosphorylating the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn activates downstream signaling pathways. PIP3 acts as a docking site for AKT, facilitating its activation through phosphorylation. Notably, one of the significant downstream AKT targets the mammalian target of rapamycin (mTOR), which plays a critical role in protein synthesis, cellular growth, and metabolic regulation. Aberrant activation of PI3K can occur through various mechanisms, including genetic mutations, PI3K genes amplification, or upstream receptors activation such as EGFR. Dysregulation of the PI3K/AKT/mTOR pathway has been implicated in various diseases, including cancer, highlighting its significance as a therapeutic target for intervention and treatment strategies (Fruman and Rommel, 2014;Polivka and Janku, 2014;Janku et al., 2018). Several inhibitors have been developed to target the PI3K/AKT/mTOR pathway. These include PI3K inhibitors such as buparlisib and sonolisib, which specifically target the PI3K enzyme and AKT inhibitors like MK-2206 and ipatasertib that block the activity of the AKT protein, a downstream effector of PI3K. Sirolimus and everolimus are targeted towards mTOR, a key component of the pathway. Additionally, dual PI3K/mTOR inhibitors such as dactolisib and apitolisib simultaneously target PI3K and mTOR.

NOTCH target
NOTCH signaling involves the activation of NOTCH receptors by ligands such as JAG1, resulting in the release of the Notch intracellular domain (NICD). The NICD then translocates to the nucleus and forms a complex with transcriptional regulators to activate target genes, including the hairy and enhancer of split (HES) family genes. This signaling pathway plays a crucial role in cell fate determination, differentiation, and various cellular processes (Siebel and Lendahl, 2017;Li et al., 2023). NOTCH receptors mAbs such as tarextumab and demcizumab, have been developed and tested in preclinical and clinical studies.

Wnt target
The Wnt signaling pathway plays a critical role in various biological processes, including embryonic development, tissue homeostasis, and cell proliferation. Dysregulation of this pathway has been implicated in several diseases, particularly cancer. Recruitment and activation of Disheveled (Dvl) are initiated by the activation of the Wnt signaling pathway. Subsequently, a complex involving Adenomatous Polyposis Coli (APC), glycogen synthase kinase 3β (GSK-3β), Casein kinase 1 (CK1), and Axin forms, leading to phosphorylation and inhibition of GSK3β (Duchartre et al., 2016;Zhang and Wang, 2020). This, in turn, results in increased levels of β-catenin, a pivotal component of the Wnt signaling pathway, which drives cancer cell proliferation. WNT974 and CGX1321, inhibitors targeting the Wnt ligand/receptor interface, have shown promising efficacy in preclinical studies (Rodon et al., 2021a;Rodon et al., 2021b).

NTRK gene fusion target
The tropomyosin receptor kinase (TRK) family comprises TRK A-C, which is encoded by neurotrophic TRK (NTRK)1-3. NTRK gene fusion occurs when the 3′region of the NTRK gene and 5′end of the fusion chaperone gene are connected by intra-chromosome or inter-chromosome rearrangement. The protein encoded by the fusion gene can bind to TRK and activate the downstream PI3K and MAPK pathways, resulting in tumor growth, proliferation, and differentiation. NTRK gene fusion was first identified in patients with CRC and then in those with other tumors (Martin-Zanca et al., 1986;Vaishnavi et al., 2015;Amatu et al., 2019;Solomon et al., 2019). Entrectinib, which targets the NTRK fusion gene, has been shown to be highly therapeutic for patients with NTRK. (2019).

Common targeted agents for CRC therapy 3.1 Bevacizumab
Bevacizumab specifically binds to VEGF, blocking its interaction with the receptor, degrading existing tumor blood vessels, normalizing surviving ones, and inhibiting tumor neovascularization to exert antitumor effects.

Adjuvant therapy
The QUASAR 2 study showed that CRC patients treated with capecitabine (Cap) + bevacizumab or only Cap after radical (R0) resection had 3-year disease-free survival (DFS) rates of 75.4% and 78.4%, respectively (Kerr et al., 2016). Similarly, the NSABP protocol C-08 and other studies (Allegra et al., 2011;de Gramont et al., 2012;André et al., 2020) confirmed that neoadjuvant chemotherapy with bevacizumab in combination with oxaliplatin did not increase DFS in patients compared to that in patients treated with chemotherapy alone. Overall, none of these trials showed a significant survival benefit for CRC patients treated with adjuvant bevacizumab; therefore, none of the current guidelines recommend the use of bevacizumab as adjuvant therapy. Adjuvant and neoadjuvant treatment trials in mCRC are summarized in Table 1.

Neoadjuvant therapy
A previous study showed that six cycles of bevacizumab in combination with chemotherapy, followed by chemoradiotherapy (CRT) achieved an objective response rate (ORR) of 88.9% in locally advanced colorectal patients with T4 or high-risk T3, with R0 resection rate of 97.8% (Masi et al., 2019). Similarly, the CRAB study reported a 95% R0 resection rate in patients with stage II/III rectal cancer treated with neoadjuvant bevacizumab + CRT (Velenik et al., 2020). Although these studies reported positive outcomes, available evidence is insufficient to support the adoption of bevacizumab as a standard neoadjuvant (Table 1).

First-line treatment
The AVF2107 study showed the administration of FOLFIRI + bevacizumab as a first-line treatment significantly improved (p < 0.001) the median overall survival (mOS; 20.3 vs. 15.6 months) and median progression-free survival (mPFS; 10.6 vs. 6.2 months) of mCRC patients compared to those of the mCRC patients treated with chemotherapy alone (Hurwitz et al., 2004). This finding facilitated the approval of bevacizumab by the United States Food and Drug Administration (FDA) as a first-line mCRC treatment in 2004. Additionally, the administration of bevacizumab + chemotherapy significantly increased (p = 0.0023) the mPFS of patients compared to that of the patients treated with chemotherapy alone (9.4 vs. 8.0 months) but did not affect mOS (Saltz et al., 2008). Moreover, both the MAVERICC (Parikh et al., 2019) and ARIES studies (Bendell et al., 2012) confirmed that FOLFIRI or FOLFOX + bevacizumab had similar PFS and OS.
However, the TRIBE2 study (Cremolini et al., 2020) showed that FOLFOXIRI + bevacizumab achieved higher mOS (27.4 vs. 22.5 months) and mPFS (19.2 vs. 16.4 months) than mFOLFOX6 + bevacizumab. The treatment benefit was independent of RAS and BRAF mutation status but was better in patients with right-sided tumors, and the same benefit was achieved in patients who progressed after treatment with FOLFOXIRI + bevacizumab (Loupakis et al., 2015;Cremolini et al., 2018b). Moreover, both the AVEX (Cunningham et al., 2013) and PRODIGE20 (Aparicio et al., 2018a) studies confirmed that bevacizumab provided increased treatment benefits in elderly patients and did not induce adverse events. Key trials of anti-VEGF agents in the treatment of mCRC are illustrated in Table 2.
Frontiers in Pharmacology frontiersin.org 04 In the CAIRO3 study (Simkens et al., 2015), bevacizumab + Cap was used as maintenance treatment for mCRC patients after first-line treatment with chemotherapy + bevacizumab that led to improved mPFS (8.5 vs. 4.1 months, p < 0.0001); however, the maintenance treatment did not affect OS. Similarly, both the PRODIGE9 (Aparicio et al., 2018b) and AIO0207 (Hegewisch-Becker et al., 2015) studies confirmed bevacizumab-induced improvement in PFS after induction chemotherapy. Moreover, the 2016 ESMO guidelines state that Cap + bevacizumab can be used for maintenance therapy after first-line treatment, but bevacizumab alone is not recommended for maintenance therapy. Additionally, the National Comprehensive Cancer Network (NCCN) guidelines do not recommend bevacizumab as maintenance therapy.

Second-line treatment
The E3200 study (Giantonio et al., 2007) showed that the administration of bevacizumab as a second-line treatment in mCRC patients achieved mOS of 12.9, 10.2, and 10.8 months and mPFS of 7.3, 2.7, and 4.7 months in patients subjected to FOLFOX + bevacizumab, bevacizumab only, or chemotherapy only first-line therapies, respectively. Thus, bevacizumab was approved in 2006 as a second-line treatment for patients with mCRC. Additionally, the ML18147 study showed that the administration of chemotherapy + bevacizumab or chemotherapy only as a second-line treatment achieved mOS of 11.2 vs. 9.6 months (p = 0.0062) and mPFS of 5.7 vs. 4.1 months (p < 0.0001), respectively, in mCRC patients subjected to bevacizumab as a first-line therapy (Bennouna et al., 2013). Moreover, the ML18147 and several other studies confirmed that retreatment with bevacizumab-containing regimens exerted significant therapeutic effects in mCRC patients who received bevacizumab-containing regimens as first-line therapy (Grothey et al., 2008;Hurwitz et al., 2014). Based on these findings, the FDA approved bevacizumab as a cross-line therapy in 2013.

Third-line treatment and beyond
A previous study showed that trifluridine and tipiracil (TAS-102) + bevacizumab was more effective than TAS-102 alone as a third-line treatment in patients with anti-EGFR positive and chemotherapy-resistant mCRC, with considerable increase in mOS (9.4 vs. 6.7 months, p = 0.028) and mPFS (4.6 vs. 2.6 months, p = 0.0010) (Pfeiffer et al., 2020). Similarly, the TAS-CC3 study confirmed the positive effects of TAS-102 + bevacizumab as a third-line treatment in Asian patients with mCRC (Yoshida et al., 2021). Accordingly, the NCCN guidelines recommend the use bevacizumab in combination with TAS-102 as a third-line treatment in patients with mCRC (Table 2).

Ziv-aflibercept
Ziv-aflibercept is an anti-VEGF agent that inhibits neovascularization by tightly binding to VEGF and reducing vascular permeability. The AFFIRM study showed (Folprecht et al., 2016) that abatacept did not achieve promising outcomes as a first-line treatment for mCRC. However, considerable therapeutic benefit was obtained with Zivaflibercept as a second-line treatment . Notably, FOLFIRI + Ziv-aflibercept was more effective than FOLFIRI only as a second-line treatment in mCRC patients, with improved mOS (13.5 vs. 12.06 months, p = 0.0032) and mPFS (6.90 vs. 4.67 months, p < 0.0001). Accordingly, Ziv-aflibercept was approved by the FDA as a second-line treatment for mCRC patients who progressed or were resistant to first-line oxaliplatin therapy in 2012 (Table 2).

Cetuximab
Cetuximab binds specifically to EGFR and competitively blocks VEGF and other receptors, inhibiting intracellular signaling pathways, thereby suppressing the proliferation of cancer cells and inducing apoptosis.

Adjuvant therapy
In the N0147 [NCCTG] study (Alberts et al., 2012), mFOLFOX + cetuximab did not show significant health-promoting effects as an adjuvant compared to those of chemotherapy alone, with no statistical difference in 3-year DFS in patients with KRAS wildtype (WT). Moreover, the PETACC-8 (Taieb et al., 2014) study confirmed that cetuximab adjuvant therapy had no survival benefit in patients with KRAS WT. Current guidelines do not recommend the use of cetuximab as an adjuvant therapy (Table 1).

Neoadjuvant treatment
The EXPERT-C study (Dewdney et al., 2012) showed that CAPOX + cetuximab was more effective than CAPOX only (four cycles each) in neoadjuvant therapy, with significantly higher response rate (RR) and OS in the cetuximab group. However, some studies, including the COIN study (Maughan et al., 2011;Tveit et al., 2012), showed that cetuximab treatment did not improve patient survival; in contrast, the TAILOR study  confirmed cetuximab OS benefit. The 2012 NCCN guidelines do not recommend cetuximab as a neoadjuvant therapy because of insufficient evidence (Table 1).

First-line treatment
The CRYSTAL study (Van Cutsem et al., 2009) showed that treatment with FOLFIRI + cetuximab significantly increased mOS (24.9 vs. 21.0 months) and mPFS (9.9 vs. 8.7 months) in patients      showed that cetuximab + FOLFOX increased mPFS (9.2 vs. 7.4 months, p = 0.004) and mOS (20.7 vs. 17.8 months, p = 0.02) in patients with RAS WT mCRC compared to those in FOLFOX alone treated patients with RAS WT mCRC. Moreover, previous studies have shown that cetuximab was as effective as bevacizumab in patients with RAS WT (Heinemann et al., 2014;Venook et al., 2017). Additionally, the NCT00208546 study showed insignificant difference in efficacy between cetuximab + bevacizumab and cetuximab only (Tol et al., 2009); therefore, cetuximab + bevacizumab is not recommended for patients with mCRC. The CALGB/SWOG80405 (Alliance) study  found that cetuximab achieved significantly higher OS and PFS in patients with left-sided primary tumors than in those with right-sided primary tumors. Several studies confirmed that anti-EGFR antibody in combination with chemotherapy exerted the best effects in patients with left-sided primary tumors and RAS WT mCRC (Arnold et al., 2017;Tejpar et al., 2017). Pivotal trials of anti-EGFR monoclonal antibody treatment in mCRC are summarized in Table 3.

Maintenance treatment
The MACRO2TTD study (Aranda et al., 2018) confirmed insignificant differences in mPFS and mOS between continuation of the original regimen and maintenance treatment with cetuximab alone after induction therapy with mFOLFOX + cetuximab. Moreover, the MACBETH study (Cremolini et al., 2018a) suggested that cetuximab could be used for maintenance therapy in patients with RAS/RAF WT mCRC. However, current guidelines do not recommend the use of cetuximab for maintenance therapy.

Second-line treatment
The BOND study (Cunningham et al., 2004) confirmed that cetuximab in combination with chemotherapy achieved a better therapeutic benefit than cetuximab alone in mCRC treatment, with increase in mOS (8.6 vs. 6.9 months, p = 0.48) and mPFS (4.1 vs. 1.5 months, p < 0.001). Cetuximab was approved by the FDA in 2004 as a second-line treatment for patients with mCRC. Subsequent studies (Sobrero et al., 2008;Ciardiello et al., 2016) have confirmed the therapeutic benefit of cetuximab in combination with chemotherapy over chemotherapy alone. Moreover, the UNICANCERPRODIGE18 study (Bennouna et al., 2019) showed that cetuximab had therapeutic benefits comparable to those of bevacizumab as second-line treatment.

Third-line treatment
The NCT00079066 study (Jonker et al., 2007) demonstrated that cetuximab monotherapy resulted in higher OS and PFS in patients with refractory mCRC than the best supportive care (BSC) alone. Vincenzi et al. (2006) treated patients with mCRC using cetuximab + irinotecan as third-line chemotherapy, with an mPFS of 4.7 months and mOS of 9.8 months.   Rechallenge therapy means the reintroduction of targeted agent to which a tumor has already proven to be resistant (Tonini et al., 2013). Santini et al. (2012) performed cetuximab rechallenge as a third-line treatment for refractory mCRC and achieved promising results, with ORR of 53.8% [partial response (PR), 48.7%; complete response (CR), 5.1%] and mPFS of 6.6 months. Similarly, patients with KRAS WT mCRC rechallenged with cetuximab as third-line treatment exhibited positive results, with mPFS and OS of 2.4 and 8.2 months, respectively (Masuishi et al., 2020). A meta-analysis conducted by Mauri et al. (2019) showed that anti-EGFR rechallenge therapy yielded better therapeutic benefits than sequential and dose escalation therapies. Therefore, cetuximab could be used as a thirdline treatment (Table 3).

Panitumumab
Panitumumab is an IgG2 monoclonal antibody that binds to EGFR, blocking the binding of VEGFR and VEGF and inhibiting cancer cell growth.

First-line treatment
The PRIME study (Douillard et al., 2010) showed that treatment with FOLFOX4 + panitumumab significantly improved mPFS in patients with KRAS WT mCRC compared to that in patients with KRAS WT mCRC treated with FOLFOX4 alone (9.6 vs. 8.0 months, p = 0.02) but did not affect OS and was not significant in patients with KRAS MT. Moreover, the ASPECCT trial  confirmed that panitumumab achieved results comparable to those of cetuximab in patients with KRAS WT mCRC. Based on these findings, panitumumab was approved by the FDA as a first-line treatment for patients with KRAS WT mCRC in 2014. A retrospective analysis of the PRIME study (Douillard et al., 2013) showed that all patients with RAS WT benefited from panitumumab treatment. Interestingly, the PEAK study (Schwartzberg et al., 2014) showed that panitumumab had better efficacy than bevacizumab in patients with KRAS/NRAS WT mCRC. Therefore, the FDA included NRAS WT mCRC as an indication for panitumumab treatment in 2017. Additionally, studies (Arnold et al., 2017;Boeckx et al., 2017;Peeters et al., 2018) have shown that panitumumab was more effective against RAS WT tumors located on the left side than those on the right side.
Furthermore, although treatment with mFOLFOXIRI + panitumumab did not significantly affect mOS and mPFS in patients with RAS WT mCRC, there was a significant increase (p = 0.004) in ORR (87.3% vs. 60.6%, p = 0.004) and metastasis resection-free recurrence survival (7.9 vs. 4.0 months) in the mFOLFOXIRI + panitumumab group compared to those in the mFOLFOXIRI group (Modest et al., 2019a). Similar results were found in the GONO study (Fornaro et al., 2013). Overall, panitumumab + FOLFOXIRI could be used for patients with RAS WT mCRC with metastases that require surgical resection.

Maintenance treatment
The NCT02476045 study (Pietrantonio et al., 2019) showed that 5-Fu + panitumumab was more effective than 5-Fu only as maintenance treatment in patients with RAS WT mCRC, with 10-month PFS of 59.9% and 49.0% (p = 0.01) in the 5-Fu + panitumumab and 5-Fu groups, respectively. Additionally, there were no significant differences in PFS, OS, and RR between panitumumab + mFOLFOX6-and 5-Fu + panitumumab-treated patients with RAS WT mCRC (Munemoto et al., 2019). Moreover, the PANAMA study (Modest et al., 2022) confirmed that 5-Fu + panitumumab was a better maintenance treatment than 5-Fu alone. A retrospective analysis of the PRIME and PEAK studies confirmed that panitumumab was comparable to bevacizumab as a maintenance therapy (Modest et al., 2019b), indicating that panitumumab could be combined with other agents as a maintenance therapy for patients with RAS WT mCRC.

Second-line treatment
The PICCOLO study (Seymour et al., 2013) showed that irinotecan + panitumumab was effective as a second-line treatment against KRAS WT mCRC than irinotecan only, as evidenced by a significant increase in PFS and RR in the panitumumab group; however, the OS was not significantly affected. Moreover, panitumumab had limited effects on patients with RAS MT. Similar results were obtained in the 20050181 study (Peeters et al., 2010;Peeters et al., 2014); therefore, panitumumab and cetuximab are sometimes used interchangeably as second-line treatments.

Third-line treatment and beyond
Cutsem Eric Van Cutsem et al. (2007) reported that panitumumab monotherapy as a third-line treatment significantly increased (p < 0.0001) mPFS to 8 weeks in patients with mCRC compared to that in patients with mCRC treated with BSC only (7.3 weeks) but did not affect OS. Accordingly, panitumumab was approved by the FDA as a third-line treatment for patients with EGFR-positive mCRC in 2006. Similar results were observed in patients retreated with panitumumab + BSC (Van Cutsem et al., 2008). Overall, several studies have confirmed the efficacy of panitumumab monotherapy as a third-line treatment for mCRC (Hecht et al., 2007;Kim et al., 2016;Kim et al., 2018). Moreover, the GERCOR (André et al., 2013) and WJOG6510G (Sakai et al., 2020) studies both confirmed that panitumumab + irinotecan improved PFS in patients with KRAS WT mCRC but did not affect OS (Table 3).

Ramucirumab
Ramucirumab exerts its antitumor effects by specifically binding to VEGFR-2 and inhibiting tumor angiogenesis. The RAISE study (Tabernero et al., 2015) showed that treatment with FOLFIRI + ramucirumab increased mOS (p = 0.0219) and mPFS (p < 0.0005) to 13.3 and 5.7 months, respectively, compared with 11.7 and 4.5 months, respectively, in FOLFIRI-treated patients. Moreover, the treatment benefit of ramucirumab was superior to chemotherapy-only for both OS and PFS in any subgroup. Ramucirumab was approved as a second-line treatment for patients with mCRC by the FDA in 2015 (Table 3).

Fruquintinib
Fruquintinib is a VEGFR inhibitor that blocks neointimal growth associated with tumor proliferation and is a potent and highly selective small-molecule inhibitor of VEGFR1-3. The Frontiers in Pharmacology frontiersin.org FRESCO study  showed that fruquintinib + BSC treatment achieved considerable therapeutic effects in mCRC patients, irrespective of whether the patients were previously treated with anti-VEGF or anti-EGFR drugs, as evidenced by an increase (p < 0.001) in mOS (9.3 months) and mPFS (3.7 months) compared to those in mCRC patients treated with the BSC only (mOS, 6.6months; mPFS, 1.8months). Accordingly, fruquintinib was approved by the China National Medical Products Administration as a third-line treatment for patients with mCRC in 2018; however, fruquintinib has not been approved for use in other countries. Similarly, the results of FRESCO-2 study showed that fruquintinib treatment significantly improved (p < 0.001) mOS (7.4 months) and mPFS (3.7 months) compared to those of BSC treatment (mOS, 4.8 months; mFPS, 1.8 months) (Dasari et al., 2022). Key trials of HER2 targeted agents and TKIs in mCRC are summarized in Table 4.

Regorafenib
Regorafenib is a TKI that acts on multiple targets, such as VEGFR and BRAF V600E. The CORRECT study (Grothey et al., 2013) showed that regorafenib + BSC improved mOS (6.4 months) and mPFS (1.9 months) in patients with mCRC compared to patients with mCRC treated with BSC only (mOS, 5 months; mPFS, 1.7 months). Accordingly, regorafenib was approved as a third-line treatment for patients with mCRC by the FDA in 2012. The CONCUR study (Li et al., 2015) demonstrated the benefits of regorafenib in Asian population. Regorafenib is the only monotherapy recommended by the NCCN guidelines for thirdline therapy and further treatment (Table 4).

Encorafenib
Encorafenib primarily targets the BRAFV600E MT, which is commonly found in certain types of cancer. Additionally, it has inhibitory effects on JNK1-3, LIMK1-4, and STK36. The BEACON study (Kopetz et al., 2019) showed that encorafenib + binimetinib + cetuximab triple-agent treatment exerted considerable therapeutic effects in mCRC patients compared to those in the mCRC patients treated with FOLFIRI + cetuximab (control). However, subsequent studies showed insignificant differences in OS and mPFS between encorafenib + binimetinib + cetuximab and encorafenib + cetuximab groups (Tabernero et al., 2021). Accordingly, the NCCN guidelines recommend the use of encorafenib in combination with anti-EGFR as a second-line treatment in patients with BRAFV600E MT mCRC (Table 4).

Dabrafenib and trametinib
Dabrafenib specifically targets BRAF mutations (V600E and V600K), while trametinib selectively inhibits MEK1 and MEK2, key components of the RAS/RAF/MEK/ERK signaling pathway, resulting in the tumor growth suppression. The NCT01072175 study (Corcoran et al., 2015) showed that combined second-line treatment with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) increased mPFS to 3.5 months in patients with BRAF V600E MT mCRC, with PR observed in 12% of the patients and CR in one patient. Similarly, the NCT01750918 study (Corcoran et al., 2018) showed that panitumumab + dabrafenib + trametinib treatment increased RR (21%), mPFS (4.2 months), and mOS (13.2 months) in patients with BRAFV600E MT mCRC compared to those in patients with The current strategies for molecular targeted therapy in mCRC.
Frontiers in Pharmacology frontiersin.org BRAFV600E MT mCRC treated with panitumumab + dabrafenib or panitumumab + trametinib. Therefore, the 2020 NCCN guidelines recommend that dabrafenib + trametinib could be combined with cetuximab or panitumumab as a second-line treatment for BRAFV600E MT mCRC. However, the 2021 and 2022 NCCN guidelines do not include dabrafenib and trametinib in combination with anti-EGFR for mCRC treatment (Table 4).

Vemurafenib
Vemurafenib is a targeted therapy that specifically inhibits the mutated BRAF form, BRAFV600E. A combination of vemurafenib and panitumumab has been shown to have a 100% tumor shrinkage rate, mPFS of 3.2 months, and mOS of 7.6 months in patients with BRAFV600E MT mCRC (Yaeger et al., 2015). Similarly, RR of 35% and mPFS of 7.7 months was obtained in patients with BRAFV600E MT mCRC treated with a combination of irinotecan, vemurafenib, and cetuximab (Hong et al., 2016). Additionally, the SWOGS1406 study (Kopetz et al., 2021) showed that irinotecan + cetuximab + vemurafenib treatment significantly increased PFS in patients with BRAFV600E MT mCRC compared to patients with BRAFV600E MT mCRC administered treatment regimens without vemurafenib. Moreover, FOLFIRI + cetuximab + vemurafenib treatment achieved an ORR of 81%, mPFS of 9.7 months, and mOS of 15.4 months in patients with BRAFV600E MT mCRC (Wang et al., 2022). Overall, these results confirmed that vemurafenib plus anti-EGFR can achieve significant efficacy in patients with BRAF MT mCRC; however, vemurafenib is yet to be approved for use in mCRC patients owing to shortage of relevant trials and limited number of enrolled patients (Table 4).

Sotorasib and adagrasib
Sotorasib and adagrasib are potent inhibitors of KRASG12C, specifically designed to target this mutation and act as antineoplastic agents. These agents selectively bind to and inhibit the mutant KRASG12C protein, offering potential therapeutic options for patients with KRASG12C MT cancers. In the phase 1 CodeBreaK100 study (Fakih et al., 2022) involving KRASG12C MT solid tumors, sotorasib treatment resulted in a median mPFS of 4.0 months and an ORR of 7.1% in the mCRC group. In the phase 2 study specifically conducted with mCRC patients, the ORR was 12.9%, with a mOS of 10.6 months and mPFS of 4.0 months (Hong et al., 2020). MRTX849 study (Klempner et al., 2022) showed that adagrasib + cetuximab mPFS was 6.9 vs. 5.6 months in adagrasib only group, and ORR was 46% vs. 19. The aforementioned studies have provided evidence of sotorasib and adagrasib efficacy in the treatment of mCRC. Nevertheless, additional clinical validation is required to further substantiate their practical clinical application.

Trastuzumab, pertuzumab, lapatinib and tucatinib
Trastuzumab and pertuzumab are monoclonal antibodies that specifically bind to different epitopes of the HER2 receptor, inhibiting HER2 signaling and enhancing immune-mediated destruction of tumor cells. In contrast, lapatinib is a TKI that targets both HER2 and EGFR receptors, effectively blocking their activation and downstream signaling pathways. The HERACLES study (Sartore-Bianchi et al., 2016) showed that trastuzumab + lapatinib treatment achieved an ORR of 30% in patients with KRAS WT and HER2-positive mCRC who previously underwent anti-EGFR therapy. Moreover, trastuzumab + pertuzumab treatment (HER2 antibodies) achieved an ORR of 32%, mPFS of 2.9 months, and mOS of 1.5 months in patients with HER2 positive mCRC (Meric-Bernstam et al., 2019) and an mPFS of 5.3 months in RAS WT subgroup. The HERACLES-B study (Sartore-Bianchi et al., 2020) showed that trastuzumab + panitumumab treatment achieved an ORR of 9.7% and mPFS of 4.1 months in patients with RAS WT and HER2-positive mCRC who had previously been treated with a third-line regimen.
Trastuzumab deruxtecan (DS-8201) is a novel antibody-drug conjugate with a humanized anti-HER2 antibody, cleavable peptide linker, and potent topoisomerase I inhibitor payload that has been confirmed to be effective in multiple solid tumors, including CRC (Tsurutani et al., 2020). The DESTINY-CRC01 study (Siena et al., 2021;Yoshino et al., 2023) treated HER2-positive mCRC patients with DS-8201 after two or more prior regimens. The patients were divided into three cohorts based on HER2 expression levels: cohort A (HER2-positive, IHC 3+ or IHC 2+/ISH+), cohort B (HER2 IHC 2+/ISH-), and cohort C (HER2 IHC 1+). The mOS in cohorts A, B, and C was 15.5, 7.3, and 7.7 months, respectively, while the mPFS was 6.9, 2.1, and 1.4 months, respectively. Notably, the ORR was observed only in cohort A, with a rate of 24%.
Tucatinib blocks proliferation and the phosphorylation of HER2 and its downstream effector, which is a TKI. MOUNTAINEER Trial (Strickler et al., 2022) rolled mCRC patients with RAS WT and HER2-positive which was treatment before but without anti-HER2, mOS was 24.1 months and mPFS was 8.2 months in the tucatinib + trastuzumab. As a result of the remarkable breakthrough in this study, the FDA granted accelerated approval in 2023 for the use of tucatinib + trastuzumab as a second-line treatment in HER2+ and RAS WT mCRC patients.
In light of the research findings mentioned above, the NCCN guidelines recommend trastuzumab in combination with either pertuzumab, lapatinib, tucatinib, or DS-8201 as second-line and beyond treatment options for patients with mCRC who have RAS WT and HER2-positive (Table 4).

Conclusion
Recent years have witnessed remarkable advancements in tumor research, driven by breakthroughs in sequencing technologies. These advancements have enhanced our understanding of tumors, including their genetic and genomic alterations. The knowledge provided a basis for personalized treatments, identification of new therapeutic targets, and improved diagnostic methods for different types of cancer. Particularly, the rapid progress in single-cell sequencing Frontiers in Pharmacology frontiersin.org technology has revolutionized the field by enabling investigations at individual tumor cell level. This approach has provided unprecedented insights into tumor heterogeneity, allowing us to gain a better understanding of the diverse cellular composition within tumors and identify potential therapeutic targets. Furthermore, it has enhanced our understanding of the tumor microenvironment, including the distinct subpopulations and functions of different cells, as well as their intricate interactions. These advancements have particularly highlighted the crucial role of the tumor microenvironment in studying mechanisms of drug resistance.
In the context of targeted therapy for tumors, significant improvements have been made in terms of CRC survival rates. The current strategies for targeted therapy in mCRC are summarized in Figure 3. However, several challenges remain. Drug resistance remains an inescapable obstacle, as the development of resistance in patients often goes unnoticed until disease progression occurs. Additionally, economic costs pose a major concern, as the need for testing multiple target markers further amplifies the financial burden on patients. Moreover, adverse drug reactions are a significant consideration, especially among vulnerable populations such as the elderly and children, given the varying tolerances to drug dosages. Striking a balance between achieving optimal efficacy and minimizing adverse effects remains an ongoing area of research.
Furthermore, the integration of immunotherapy and molecular targeted therapy has shown potential for further improvement of the survival rates of patients with tumors, including those with mCRC. However, combination of these therapies presents its own set of challenges requiring careful consideration. Co-administration of multiple drugs can potentially intensify adverse reactions, underscoring the importance of identifying the most effective combination regimen through thorough evaluation.
In summary, scientific progress has significantly enhanced our ability to combat CRC and other tumors. While obstacles and unanswered questions remain, the field of targeted therapy continues to advance, and it is anticipated that future clinical trials and research efforts will yield major breakthroughs, further increasing the survival rates and overall outcomes for cancer patients.

Author contributions
SH and JY drafted the manuscript, tables and figures. XG, XH, CL assisted the literature search. RL and YaL assisted the revision. ML, LL, YoL, and JH advised on the manuscript. YaL and RL supervised and finalized the work. All authors contributed to the article and approved the submitted version.