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Developing therapeutic resistance to monoclonal antibodies (mAbs) causes increasing failure in oral squamous cell carcinoma (OSCC) treatment. A clear understanding of the molecular basis for drug resistance will pave the way for OSCC management and a new effective therapeutic modality. This rentryview elucidates the role played by EMT during the emergence of mAbs resistance and the configuration of the tumor microenvironment. The cancer cells that undergo the EMT process also cause significant energy substrate consumption which leads to a limited number and function of effective T-cells, eventually leading to immune evasion. This entryreview firstly reveals the implicit crosstalk between the EMT, energy metabolism, and therapeutic resistance of mAbs. A focus on the rebalanced energy homeostasis in cancer cells and T-cells may provide a new perspective on the treatment of OSCC.
- oral squamous cell carcinoma
- chemotherapeutic monoclonal antibodies
- cetuximab
- pembrolizumab
- nivolumab
- epithelial–mesenchymal transition
- tumor microenvironment
1. Introduction
Oral squamous cell carcinoma (OSCC), the main pathological type of oral cancer (OC), accounts for at least 90% of cases of oral malignancy [1]. Although there have been many developments in cancer treatment, the overall 5-year survival rate for OSCC remains low, at ~50–60% [2]. The standard treatments for OSCC are surgery, radiotherapy, and chemotherapy. Unfortunately, adjuvant therapies such as chemotherapy do not achieve satisfactory results, largely due to low target selectivity, severe side effects, and drug resistance. Numerous studies have demonstrated that cancer cells tend to develop chemoresistance after frequent use of high-dose chemotherapy [3].
Several phenomena, including the epithelial–mesenchymal transition (EMT) and the accumulation of cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs), are implicated in chemoresistance in OSCC [4]. The EMT is a common feature of various types of cancer and is closely associated with CSCs and CAFs. During the EMT, a change occurs in the molecular pathways of cancer cells, and gene reprogramming induces their proliferation and invasiveness. Invasive cancer cells acquire mesenchymal features by gradually losing cell polarity, tight intercellular junctions, and the ability to express epithelial markers [5]. Numerous EMT-activating transcription factors (EMT-ATFs) participate in this process, including Snail, TWIST, and ZEB. The EMT-ATFs specifically bind to the promoter of E-cadherin through E-boxes and eventually suppress the expression of the adherent junction protein E-cadherin [6]. The balance between the epithelial marker E-cadherin and mesenchymal markers plays a pivotal role in the dynamic EMT process, which affects subsequent cancer cell migration, metastasis, immune evasion, and resistance to chemotherapeutic agents [7].
While primary tumor cells undergo EMT, those cells create an optimal tumor microenvironment (TME) by secreting various cytokines and proteases to facilitate their survival and ability to evade the immune system. Stromal cells, which are affected by the TME, are also activated to release several factors that trigger the EMT in primary tumor cells [8]. Methods to inhibit the EMT and convert the TME into a normal stromal cell microenvironment are needed.
Another problem for conventional chemotherapy is that cancer cells share similarities with normal host cells, so it is difficult for therapeutic agents to achieve high levels of selective cytotoxicity. However, in the last decade, monoclonal antibodies (mAbs) have shown great potential for use against many hematological and solid cancers in humans [9]. The mAbs target cancer cells by specifically binding to cell surface antigens that are necessary for cell proliferation or differentiation, or for immune meditation. Examples of such antigens include the epidermal growth factor receptor (EGFR), programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and vascular endothelial growth factor and its receptor (VEGF and VEGFR, respectively) [10][11][12][10,11,12].
According to the National Comprehensive Cancer Network, only three mAbs, cetuximab, pembrolizumab, and nivolumab, have been used as evidence-based therapeutic regimens of mAbs for treating OSCC [13]. Although this new therapeutic approach offers many advantages, it also has several limitations, including adverse effects and the development of resistance, in addition to the high cost of producing the antibodies [14]. The development of resistance against mAbs, also seen in some OSCC patients, particularly when used alone without other chemotherapeutic agents, remains problematic [15]; nevertheless, the engineering of mAbs represents a major milestone in cancer therapeutics.
2. Cetuximab: A mAb and EGFR Inhibitor
2.1. Rapid Response of Cancer Cells to Cetuximab Treatment
Cetuximab, an IgG1 mAb, is an inhibitor of EGFR. It is used for the treatment of locoregionally advanced, metastatic, and recurrent cancers, including OSCC [16]. Compared to EGF, cetuximab has a 5-fold higher affinity for EGFR, so can bind to EGFR prior to EGF and block the interaction of the ligand with its receptor [17].
EGFR, also known as ErbB1 or HER1, is a transmembrane tyrosine kinase receptor that binds to the EGF family of extracellular protein ligands. EGF stimulates OSCC cells to undergo the EMT. Up to 90% of OSCC cells overexpress EGFR. Upregulated EGFR is associated with poor prognosis, chemoresistance, and radioresistance [18]. Cetuximab seemed to be a reasonable therapeutic strategy for OSCC, however, very few patients responded to the treatment, and virtually all responders developed resistance within a short period [19].
Based on the work of Kagohara et al. [20], a cetuximab-treated cell line developed an early transcriptional response (within one day), and EGFR inhibition therapy induced a significant transcriptional variation within five days compared to untreated cells. EGFR inhibition therapy disrupts the homeostasis of cancer cells; therefore, the cells spontaneously progress to a state with activated heterogenetic hallmark pathways to overcome EGFR inhibition. This could explain the immediate upregulation of vimentin seen when cells were treated with cetuximab [20].
As well as vimentin, other EMT-ATFs such as LEF1, TWIST1, and ZEB1 were significantly increased after treatment with cetuximab. Gene analysis of the same patients showed that Wnt, TGF-β, and NOTCH1 pathway-related genes were significantly upregulated following treatment with cetuximab. All of those genes were specifically involved in the extracellular matrix organization, including cell adhesion, migration, and the inflammatory response [21]. Five CAFs-associated genes, CXCL12, ACTA2, FAP, PDGFRB, and S100A4, were also significantly upregulated in the cetuximab-treated group compared to the control group. CXCL12, also known as stromal cell-derived factor-1α, binds to its receptor CXCR4 and activates the CXCR4/CXCL12 axis; this leads to tumor proliferation, vascularization, and metastasis [22]. Activation of the CXCR4/CXCL12 axis has been shown to decrease the levels of E-cadherin and increase those of vimentin and N-cadherin (Figure 1). These findings imply that cetuximab may induce the EMT indirectly, as well as CAFs proliferation, leading to the development of cancer cell resistance. The CXCR4 is expressed in multiple cell types, including lymphocytes, hematopoietic stem cells, epithelial cells, and cancer cells [23].
2.2. Cetuximab Mainly Targets OSCC with Epithelial Features
In one study, cetuximab failed to block EGF-driven mesenchymal progression in transformed OSCC [24][27], suggesting a novel drug resistance mechanism. An earlier study reported that esophageal squamous cell carcinoma (ESCC) cells with mesenchymal features displayed resistance not only to cetuximab but also to tyrosine kinase inhibitors (TKIs) such as erlotinib, albeit that both drugs were highly effective against ESCC cells with epithelial features [25][28].
The progression of OSCC is always associated with the release of extracellular vesicles (EVs) from cancer cells into the milieu. The EVs usually contain CD9 and EGFR [26][29]. Earlier research showed that CD9-positive EVs were secreted more often by OSCC than epithelial cells [27][30].
2.3. Combination Treatment with Cetuximab
An effective regimen for refractory OSCC is the combination of cetuximab and cisplatin. Several studies have demonstrated that this combination is beneficial in terms of the tumor response and survival rate compared to single therapeutic agents [28][29][32,33]. It was proposed that cetuximab initiates cytoplasmic signaling through the autophosphorylation of intracellular domains [30][34]. Phosphorylated EGFR activated the MAPK and PI3K/AKT/mTOR pathways, which are also involved in the therapeutic effects of cisplatin [31][35]. However, cisplatin also induces cell apoptosis through the activation of the mitochondrial signaling pathway and AMP-activated protein kinase signaling pathway (AMPK) [32][36]. AMPK is a serine/threonine kinase that regulates intracellular energy homeostasis. The activation of the AMPK pathway could rapidly reprogram glucose metabolism, inducing cells to switch from active ATP consumption to ATP production to restore energy, potentially reversing the Warburg effect [33][37].3. Immune Checkpoint Inhibitors
3.1. PD-1-Blocking Agents: Pembrolizumab and Nivolumab
Pembrolizumab was developed to block the interaction of PD-1 with its ligands and inhibit immune evasion. Pembrolizumab is a highly selective monoclonal IgG4 isotype antibody that impedes inhibitory signals in T cells [34][50]. This antibody was first approved in the United States in 2014 [35][51] as a first-line treatment for metastatic bladder cancer patients with high levels of PD-L1 and resistance to cisplatin-based treatment. In patients with OSCC/head and neck squamous cell carcinoma (HNSCC), pembrolizumab is used as a second-line treatment after platinum-based chemotherapy.
Nivolumab is another human IgG4 PD-1 immune checkpoint inhibitor antibody that blocks PD-L1 by binding to PD-1 [36][52]. Both pembrolizumab and nivolumab have been approved for patients with recurrent/metastatic OSCC who have previously undergone platinum-based chemotherapy [37][53].
PD-1 binding of pembrolizumab is dependent on a flexible C’D loop, while nivolumab targets PD-1 via the N-terminal extension and FG and BC loops (Figure 2). In a previous study of the crystal structure of pembrolizumab and nivolumab, the epitope region of the former was shown to have a much great affinity area to the PD-1 binding site than the latter [38][55]. Intriguingly, the binding sites of these two drugs on the PD-1 molecule do not overlap. These results imply that the two antibodies could act synergistically, thus, raising the question of whether they should be co-administered. Preclinical and early clinical data indicate that pembrolizumab and nivolumab can be used interchangeably [39][56].
3.2. EMT-ATFs Participate in the Configuration of an Immunosuppressive TME
A few patients initially responded to PD-1 blockade therapy but later became unresponsive, possibly due to insufficient effector T cells [40][58]. In another study, patients were unresponsive from the start of the treatment [41][59]. A recent study reported that in lung adenocarcinoma, the levels of multiple immune checkpoint molecules, including PD-L1, were increased, reflecting an EMT phenotype. This suggested that EMT could be associated with immunosuppressive TME changes [42][43][60,61]. The EMT-ATFs not only interact with the regulatory networks of microRNAs to promote cancer cell plasticity and maintain cancer stemness but also play an essential role in configuring the TME [44][62].
3.3. Interactions among Energy Metabolism, Immunosuppression, and the TME
In addition to the EMT-ATFs, a complex regulatory network consisting of noncoding RNAs, epigenetic modifiers, post-translational regulators, and alternative splicing factors has been implicated in the dynamic regulation of the TME [45][46][69,70]. All of these extracellular factors promote a hypoxic, acidic, and inflammatory TME. Immune cells in the TME secrete cytokines, inflammatory factors, and chemokines to drive the EMT in cancer cells. In turn, cancer cells interact with immune cells to promote cell plasticity and the release of immunosuppressive substances or cytokines, which induce the infiltration of Treg cells or polarized M2 macrophages into the TME; therefore, an immunosuppressive microenvironment is generated, promoting cancer genesis and therapeutic resistance. Cancer cells predominantly undergo glycolytic metabolism, which facilitates the rapid generation of ATP and inevitably leads to the accumulation of lactic acid in the milieu. T cells are similar to cancer cells in terms of their ability to recognize tumor antigens. To meet the bioenergetic demand for rapid proliferation, T cells also switch to glycolytic energy metabolism. Distinct from cancer cells, T cells produce pyruvic acid rather than lactic acid as the byproduct of glycolysis [47][71]. In accordance with the similar proliferation profile between cancer and T cells, the cells compete for energy sources and substrates for anabolic pathways. Several studies have demonstrated that glucose and tryptophan were excessively consumed by tumor cells, thereby restricting T cell glycolytic metabolism, activation, and proliferation, which eventually led to T cell dysfunction or depletion [48][49][72,73]. As a consequence, infiltration of T cells into the tumor mass was severely reduced. Furthermore, there was a decrease in the nuclear factor of activated T cells and natural killer (NK) cells, and in IFN-γ in the TME. These changes permitted cancer cells to escape from antitumor immunity [50][74]. Energy metabolism is mainly regulated by two completely opposing energy sensors: AMPK and mTOR [51][75]. The AMPK pathway regulates mitochondrial catabolism under restricted energy and nutrient conditions. However, the mTOR pathway (mainly mTOR complex 1, mTORC1) preferentially triggers anabolic metabolism [52][53][76,77]. Zhang et al. revealed that mTORC1 inhibitors activated the AMPK pathway, suppressed the EMT process, and downregulated PD-L1 expression in lung cancer [54][78]. In another report, the upregulated expression of PD-L1 was observed predominantly in patients with K-RAS mutations [55][79]. The overexpression of RAS promoted the progression of EMT by decreasing E-cadherin and increasing the expression of TGFβ, vimentin, and Snail, most likely via the MAPK and PI3K/AKT/mTOR pathways in concert with mTORC1.45. A potential Novel Combination Therapy
4.1. Combined Use of the Small-Molecule Inhibitors MEK
5.1. Combined Use of the Small-Molecule Inhibitors MEK
Blocking the PI3K/AKT/mTOR and MAPK pathways by small-molecule inhibitors may be effective for treating OSCC. To date, four small-molecule MEK inhibitors have been approved by the FDA: trametinib, selumetinib, cobimetinib, and binimetinib [56][101]. Several other inhibitors are also under development, such as PD184352 [57][102] and PD0325901 [58][103].
The combination of a MEK inhibitor and immunotherapy was associated with a satisfactory toxicity profile in BRAFV600E-mutant melanoma patients [59][60][106,107]. In a phase II trial of the triple combination of trametinib, dabrafenib, and pembrolizumab, the objective response rate was 63.3% (38 out of 60 patients with BRAFV600E-mutated advanced melanoma); 17 of these 38 patients (44.7%) had ongoing responses [61][108].
4.2. EGFR and PD-1 Inhibitors
5.2. EGFR and PD-1 Inhibitors
45.2.1. EGFR-TKIs with a PD-1 Inhibitor
Preclinical studies indicated that the activation of EGFR not only stimulated tumor growth but was positively correlated with the overexpression of PD-L1. Treatment of EGFR-mutant non-small cell lung cancer (NSCLC) cell lines with EGFR-TKIs decreased the expression of PD-1 and PD-L1 by inhibiting the NF-κB pathway [62][63][64][109,110,111]. These studies provided a theoretical basis for the combination treatment of EGFR-TKIs and immunotherapy. Haratani et al. reported that T790-negative patients with EGFR mutation-positive NSCLC were more likely to benefit from nivolumab after EGFR-TKI treatment [65][112]. This result was attributed to the fact that NSCLC patients with certain types of EGFR mutations may have a higher nonsynonymous mutation burden, such that they are more responsive to PD-1 inhibitors. However, it should be noted that the trial mainly included lung cancer patients rather than OSCC patients. OSCC is characterized by a higher tumor mutation burden, higher expression of PD-L1, and increased CD8+ T cell infiltration; therefore, OSCC patients can be expected to benefit more from combination treatment.
45.2.2. EGFR-mAb with a PD-1 Inhibitor
A multi-center phase II study investigating the combination of the EGFR-mAb cetuximab and PD-1 inhibitor pembrolizumab was conducted from 2017 to 2019. The study focused on recurrent and metastatic HNSCC; in 45% of patients, the primary tumor site was in the oral cavity [66][113]. An overall response rate of 45% was achieved with combination therapy, which exceeded the response rates for pembrolizumab (16–18%) and cetuximab (6–13%) monotherapy [67][114]. We suggest that the resistance of cancer cells to cetuximab may be associated with a K-RAS mutation. The activation of the MAPK pathway enhanced the expression of PD-L1 and the infiltration of T cells and, thus, the response to immunotherapy. Although its mechanism of action is yet to be fully elucidated, this combination therapy has a promising future.