Edited by: Nurit Hollander, Tel Aviv University, Israel
Reviewed by: Renee Donahue, National Institutes of Health (NIH), United States; Fermín E. González, University of Chile, Chile; Fabiola Osorio, University of Chile, Chile
This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology
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Although melanoma remains the deadliest skin cancer, the current treatment has not resulted in the desired outcomes. Unlike chemotherapy, immunotherapy has provided more tolerable approaches and revolutionized cancer therapy. Although dendritic cell-based vaccines have minor side effects, the undesirable response rates of traditional approaches have posed questions about their clinical translation. The immunosuppressive tumor microenvironment can be the underlying reason for their low response rates. Immune checkpoints and indoleamine 2,3-dioxygenase have been implicated in the induction of immunosuppressive tumor microenvironment. Growing evidence indicates that the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Protein kinase B (PKB) (PI3K/AKT) pathways, as the main oncogenic pathways of melanoma, can upregulate the tumoral immune checkpoints, like programmed death-ligand 1. This study briefly represents the main oncogenic pathways of melanoma and highlights the cross-talk between these oncogenic pathways with indoleamine 2,3-dioxygenase, tumoral immune checkpoints, and myeloid-derived suppressor cells. Moreover, this study sheds light on a novel tumor antigen on melanoma, which has substantial roles in tumoral immune checkpoints expression, indoleamine 2,3-dioxygenase secretion, and stimulating the oncogenic pathways. Finally, this review collects the lessons from the previous unsuccessful trials and integrates their lessons with new approaches in RNA-modified dendritic cell vaccines. Unlike traditional approaches, the advances in single-cell RNA-sequencing techniques and RNA-modified dendritic cell vaccines along with combined therapy of the immune checkpoint inhibitors, indoleamine 2,3-dioxygenase inhibitor, and RNA-modified dendritic cell-based vaccine can overcome these auto-inductive loops and pave the way for developing robust dendritic cell-based vaccines with the most favorable response rate and the least side effects.
Melanoma is the malignant proliferation of neural-crest-derived pigment-producing cells located in the skin, inner ear, eye, and leptomeninges (
Since dendritic cells (DCs) can bridge innate and adaptive immunity, they have focal roles in developing anti-tumoral immune responses (
The transmembrane glycoprotein mucin 1 (MUC1) is a novel tumoral antigen on melanoma cells (
Recent studies have demonstrated multiple interplays between melanoma oncogenic pathways, MUC1, the abovementioned tumoral immune checkpoints, MDSCs, and indoleamine 2,3-dioxygenase (IDO). These auto-inductive loops can inhibit the development of anti-tumoral immune responses in the melanoma microenvironment. Therefore, targeting these loops can bring ample opportunity to improve the response rates of DC-based vaccines in affected patients. Furthermore, recent advances in single-cell RNA-sequencing techniques and engineered DC-based vaccines have furthered our knowledge of tumor biology and provided ample opportunity to develop potent DC-based vaccines. Identifying new biomarkers along with the previously established tumor-related antigens and genetic modification of DC vaccines might be a promising approach for the treatment of melanoma patients. This study aims to highlight the cross-talk between the main oncogenic pathways of melanoma and immunosuppressive inducer factors, i.e., tumoral immune checkpoints, MDSCs, and IDO. This study also intends to collect lessons from the RNA-modified DC vaccine studies and previous preclinical studies to improve the response rate of DC-based vaccines in melanoma patients.
A better understanding of melanoma transformation from extracellular and intracellular view is essential for developing a potent DC-based vaccine for melanoma patients. In the following sections, we discuss the main oncogenic signaling pathways of melanoma and their associations with the tumor microenvironment.
Extracellular signals can initiate the MAPK pathway via binding to receptor tyrosine kinases (RTKs). The stimulation of RTKs leads to rat sarcoma (RAS) activation, the membrane-bound GTPase (
The NRAS, the isoform of the RAS superfamily, is another oncogenic mutant in 30% of melanoma (
The ERK stabilizes dual-specificity phosphatases 5, which represses the ERK signaling pathway (
Two main intracellular carcinogenic pathways of melanoma and their inhibitors.
The PI3K/AKT/mechanistic target of rapamycin (mTOR) pathway is another crucial pathway in melanoma development. RTKs and activated Ras can lead to the PI3K activation. Afterward, the activated PI3K stimulates the AKT (
Since the mTOR can repress the unc-51 like autophagy activating kinase 1 (UKL1) and UKL2, it can inhibit autophagy (
Overall, these preliminary findings have raised the notion that suppressing these pathways can eliminate melanoma development; however, the subsequent adverse side effects have posed daunting challenges for this approach (see below).
The tumor microenvironment has substantial roles in determining the fate of cancer cells. Indeed, the balance between the stimulatory and inhibitory signals can determine the direction of anti-tumoral immune responses against tumor antigens. Immune checkpoints are the well-established inhibitory axes that can repress the T-cell mediated anti-tumoral immune responses and promote tumor growth (
The PD-L1/PD-1 axis is one of the well-known immune checkpoint axes. The PD-1, as a transmembrane protein, can be expressed on the T-cells and natural killer cells (
Although tumoral PD-L1 expression has been shown to promote cancer development, higher PD-L1 expression has not been correlated with inferior overall survival and progression-free survival in melanoma patients (
The cross-talk between tumoral PD-L1 and the MAPK and PI3K/AKT might be one reason for impeding anti-tumoral immune responses despite the continuous tumor growth and progression. Atefi et al. have reported that the activation of the PI3K/AKT signaling pathway can increase the expression of tumoral PD-L1, resulting in tumor development (
Besides melanoma, abundant studies reveal the correlation between the MAPK-PI3K/AKT oncogenic pathways and PD-L1 expression. In lung adenocarcinoma cells, the activation of the MAPK pathway upregulates the tumoral PD-L1 expression. Moreover, the inhibition of the MAPK pathway downregulates PD-L1 without affecting the major histocompatibility complex (MHC) class I level (
Represents the multiple positive-loops between programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), mucin 1 (MUC1), myeloid-derived suppressor cell (MDSC), dendritic cell (DC), T-cell, the MAPK signaling pathway, and the PI3K/AKT pathway. The immunosuppressive microenvironment is partially due to these unfavorable auto-inductive loops. The PD-L1/PD-1 axis is a well-established culprit of impeding anti-tumoral immune response development. The CTLA-4/CD80, CD86, another immune checkpoint axis, is responsible for suppressing the anti-tumoral immune response. The PD-L1 on the tumoral cell surface can impede the development of the anti-tumoral immune response and stimulated the PI3K/Akt and MAPK signaling pathways. MUC1 has the focal role in the recruitment of MDSC and the propelling of the MAPK pathway. The MAPK signaling pathway is also responsible for the overexpression of MUC1. Thus, these positive loops can substantially induce an immunosuppressive micro-environment. Therefore, these two major recognized signaling pathways can induce tumorigenesis and shield the tumoral cells from anti-tumoral immune responses.
TLA-4 was initially shown on the surface of T-cells. However, recent findings have identified them on the tumoral cells, e.g., melanoma and luminal B breast cancer cells (
Besides the expression of CTLA-4 on immune cells, the CTLA-4 can also be highly expressed on the cell surface of human melanoma cells (
Since tumor-related antigens are not present overtly on the non-tumoral cells, immunotherapy can efficiently target these aberrant antigens (
Although normal epidermal cells do not express MUC1, normal MUC1 can defend against infective pathogens in the digestive tract (
IDO can transform the pro-inflammatory tumor microenvironment into an immunosuppressive tumor micro-environment. Tumoral cells increase IDO level, leading to the inhibition of effector T-cells (
MDSCs can also contribute to the IDO upregulation, and in return, IDO facilitates the recruitment of MDSCs. Moreover, the PI3K/AKT signaling pathway can pave the way for recruiting MDSCs in the tumor microenvironment (
Although recent findings have indicated that there is a synapse between various DCs to share tumor antigens (
PDCs can impede the anti-tumoral immune responses of killer (NK) cells and T-cells via PD-L1 overexpression (
CDC1 is assigned to deliver antigens to the lymph node, recruit the effector T-cells via expression of chemokine (C-X-C motif) ligand 9 (CXCL9) and C-X-C motif chemokine ligand 10 (CXCL10), and stimulate T-cells and NK cells via secretion of IL-12 (
Although cDC2 is mainly responsible for the activation of CD4+ T cells, recent data have shown that cDC2 is also involved in priming CD8+ T-cells (
MoDCs are commonly developed following inflammation and can promote Th-1 and Th-17 development (
The anti-tumoral vaccines aim to expand the specific effector lymphocytes in the tumoral microenvironment. These clonal T-cells efficiently detect the specific targeted tumor-related antigen and develop an anti-tumoral immune response. DCs, as the professional APC, are the pivotal cells in the activation of specific T-cells (
The fundamental concept in developing DC-based vaccines was the training of DCs with tumoral antigen in
The application of the single-cell RNA-sequencing techniques might be a promising approach to classify the tumoral antigens and the tumor microenvironment (
Several techniques have been developed to express the desired peptide-MHC complex in DCs via mRNAs, i.e., electroporation, lipid-mediated transfection, and mRNA co-incubation, which are reviewed elsewhere (
The first DC-based vaccine for cancer was approved in 2010 for prostate cancer patients (
Another plausible target might be silencing IDO in DCs. Flatekval et al. have shown that loading IDO-siRNA can substantially improve the stimulation of T-cells (
The following sections aim to collect the learned lessons from successful and unsuccessful clinical trials. Based on these lessons and the results of preclinical studies, we will present our proposed strategy for treating melanoma patients.
The two main oncogenic pathways of melanoma, i.e., the MAPK and PI3K/AKT pathways, were the center of efforts to overcome melanoma. However, chemotherapy against these pathways has not provided well-tolerable treatment for affected patients. Due to the proliferative and anti-apoptotic ability of the MAPK signaling pathway, inhibitors targeting this signaling pathway were developed; however, these inhibitors failed to fulfill the desired outcomes because of the complexity of melanoma pathways. Vemurafenib is the first generation of BRaf inhibitor, which has passed the phase I trial with an 81% response (
The PI3K/AKT signaling pathway is another main pathway of melanoma development. Therefore, developing the PI3K/AKT pathway inhibitors was another appealing approach for treating melanoma patients. However, there have been serious issues with the efficacy of the PI3K/AKT pathway inhibitors. Although preclinical studies have demonstrated the desired outcomes in treating melanoma cells with CCI-779 mTOR kinase inhibitor, its phase II trial has revealed poor outcomes about the application of this agent (
Aberrant MUC1, which is substantially expressed in melanoma cells, has been implicated in the induction of immunosuppressive tumor microenvironment (
Although MUC1 loaded-DC vaccines have shown beneficial outcomes for patients with non-small cell lung cancer (
The immunosuppressive tumor microenvironment of melanoma inhibits the development of anti-tumoral immune responses. The overexpression of PD-L1 and CTL-A4 on the tumoral cells shields the tumoral cells from immune responses. Based on recent meta-analyses, there has been a remarkable association between tumor-infiltrating lymphocytes and tumoral PD-L1 expression in triple-negative breast cancer and melanoma (
Although most studies investigate the inhibition of PD-1 in cancer contexts, durvalumab and avelumab are novel agents, which can block tumoral PD-L1. The combination of tremelimumab, as an inhibitor of CTLA-4, and durvalumab have demonstrated promising results for patients with overexpressed PD-L1 solid tumors. However, the combination of durvalumab with tremelimumab needs a close follow-up because of the increased incidence of adverse events in treated patients (
Following the CTLA-4 and its inhibitory effect on the immune response, the first monoclonal antibody targeting CTLA-4 was introduced. However, ipilimumab was only beneficial on a small portion of patients with melanoma (
Since IDO can also be expressed from tumoral cells (
Some of the clinical trials were designed for patients with melanoma.
DC-vaccine | Biological anti-tumoral immunity | Phase II | 2002 | Terminated | NCT01042366 |
DC-vaccine | Biological anti-tumoral immunity | Phase I | 2005 | Completed | NCT00125749 |
Binimetinib and Encorafenib | Inhibition of the MAPK pathway and BRaf gene, respectively | Phase II | 2020 | Not yet recruiting | NCT04221438 |
Pembrolizumab and Ipilimumab | Inhibition of PD-1 axis and CTLA-4 axis | Phase II | 2019 | Active, not recruiting | NCT03873818 |
Nivolumab and Ipilimumab | Inhibition of PD-1 axis and CTLA-4 axis | Phase II | 2016 | Active, not recruiting | NCT02970981 |
Pembrolizumab, Cyclophosphamide, and DC-based vaccine | Inhibiting PD-1/PD-L1 axis, inhibiting protein synthesis, and Biological anti-tumoral immunity | Phase I | 2017 | Recruiting | NCT03092453 |
Standard of care immune checkpoint inhibitors and DC-based vaccine | Inhibiting immune checkpoints and inducing biological anti-tumoral immunity | Phase I/II | 2016 | Active, not recruiting | NCT02678741 |
Developing agents to suppress the oncogenic pathways had been the cornerstone of efforts to treat cancer patients. However, their side-effects and the heterogeneous nature of tumor cells have posed daunting challenges for chemotherapy. On the other hand, immunotherapy has been revolutionizing cancer therapy. Despite the low response rate of traditional peptide-based DC vaccines, the novel RNA-modified DC vaccines have shown low toxicity and promising results in preclinical studies. Loading coded mRNA for desired antigens and siRNA for targeting inhibitory axes might be a promising approach in developing DC vaccines. The data from the single-cell RNA-sequencing, along with the well-established tumoral antigens like MUC1, can help us design the desired mRNA in heterogeneous and ever-progressing cancers. The immunosuppressive tumor microenvironment of melanoma might owe to the fact that there are multiple auto-inductive loops between tumoral expressed immune checkpoints, oncogenic signaling pathways, IDO, and immunosuppressive cells. Indeed, combination therapy of immune checkpoint inhibitors and IDO might transform the immunosuppressive tumor microenvironment into the pro-inflammatory tumor microenvironment. The target antigens for gene-modified DC vaccines can be provided from the data of single-cell RNA-sequencing and previous well-known pro-tumoral tumor antigens, e.g., MUC1. Thus, this combination therapy and the administration of RNA-modified DC vaccines can suppress tumor development and provide long-lasting immunity against tumor antigens.
MS: the first author of the manuscript, collected the data, and wrote the primary version of the manuscript. KH, AD, and NS: contributed to English editing and revision of the manuscript, and also helped with data categorization. BB and VR: the corresponding authors of the manuscript contributed to writing the main text of the manuscript and supervised the manuscript. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.