Receptor Tyrosine Kinases Amplified in Diffuse-Type Gastric Carcinoma: Potential Targeted Therapies and Novel Downstream Effectors

Simple Summary Diffuse-type gastric carcinoma (DGC) is an aggressive subtype of gastric carcinoma with an extremely poor prognosis due to frequent peritoneal metastasis and high probability of recurrence. Its pathogenesis is poorly understood, and consequently, no effective molecular targeted therapy is available. The importance of oncogenic receptor tyrosine kinase (RTK) signaling has been recently demonstrated in the malignant progression of DGC. In particular, RTK gene amplification appears to accelerate peritoneal metastasis. In this review, we provide an overview of RTK gene amplification in DGC and the potential of related targeted therapies. Abstract Gastric cancer (GC) is a major cause of cancer-related death worldwide. Patients with an aggressive subtype of GC, known as diffuse-type gastric carcinoma (DGC), have extremely poor prognoses. DGC is characterized by rapid infiltrative growth, massive desmoplastic stroma, frequent peritoneal metastasis, and high probability of recurrence. These clinical features and progression patterns of DGC substantially differ from those of other GC subtypes, suggesting the existence of specific oncogenic signals. The importance of gene amplification and the resulting aberrant activation of receptor tyrosine kinase (RTK) signaling in the malignant progression of DGC is becoming apparent. Here, we review the characteristics of RTK gene amplification in DGC and its importance in peritoneal metastasis. These insights may potentially lead to new targeted therapeutics.


Introduction
Gastric cancer (GC) has the fifth-highest incidence among cancers, and it is the fourth leading cause of cancer-related death worldwide [1]. According to Lauren's classification, GC is histologically categorized into two main subtypes: intestinal-type gastric carcinoma (IGC) and diffuse-type gastric carcinoma (DGC) [2]. DGC is an aggressive subtype with an extremely poor prognosis owing to rapid infiltrative invasion within the submucosa and frequent occurrence of peritoneal dissemination and high probability of recurrence [3]. DGC contains poorly differentiated and signet ring carcinoma cells that solitarily exist within a dense tumor stroma because of the lack of cell-cell adhesion. Scirrhous gastric carcinoma (SGC), also referred to as linitis plastica, is a subtype of DGC that is characterized by an extensive desmoplastic reaction [4].
Peritoneal metastasis is the dissemination of cancer to the peritoneum, which covers the abdominal cavity and the intra-abdominal organs, and it is often associated with the formation of malignant ascites. The quality of life and survival rate of the patients are both greatly reduced by peritoneal metastasis. However, no effective molecular targeted therapy is currently available. Because the pathological and histological features of DGC are quite different from those of IGC, studies focusing on DGC are necessary to understand its molecular basis and to develop effective molecular targeted therapy. The importance of oncogenic receptor tyrosine kinase (RTK) signaling has been recently demonstrated in the malignant progression of DGC. In particular, RTK gene amplification appears to accelerate peritoneal metastasis, and thus it is considered a promising therapeutic target. In this review, we provide an overview of RTK gene amplification in DGC and the potential of related targeted therapies. The recent literature regarding the downstream effectors of RTKs is summarized. Finally, we highlight issues that should be addressed to effectively target amplified RTK in DGC.

Gene Amplification of RTKs in DGC
RTKs are cell surface receptors that regulate various cellular processes, including proliferation, survival, metabolism, differentiation, migration, and invasion. Upon binding to specific ligands, two RTK molecules dimerize and tyrosine-phosphorylate each other, and recruit and activate a variety of intracellular signaling molecules to trigger the activation of downstream signaling pathways, such as Ras/MAPK, PI3-kinase/Akt, JAK/Stat, and NF-κB pathways ( Figure 1). RTKs are well-known oncogenic drivers that are aberrantly activated in a wide range of human cancers. Oncogenic activation of RTK signaling is caused in several ways, including overexpression, activating mutations, fusions, and ligand dysregulation [5]. RTK overexpression is commonly caused by gene amplification that provokes ligand-independent dimerization owing to local enrichment, causing constitutive activation of downstream signaling pathways. Cancer cells harboring gene amplification of RTKs often exhibit 'oncogene addiction', and their growth and survival are highly dependent on the activity of amplified RTKs. Hence, RTKs amplified in cancers have been considered promising therapeutic targets; many RTK inhibitors have been developed, evaluated in preclinical and clinical trials, and used in the clinic. The amplification of RTK genes has been identified in GC, including EGFR encoding epidermal growth factor receptor (EGFR), ERBB2 encoding human epidermal growth factor receptor 2 (HER2, also known as ERBB2), MET encoding Met (also known as c-Met), and FGFR2 encoding fibroblast growth factor receptor 2 (FGFR2), as summarized in Table 1. We also performed gene-amplification analysis of these RTKs in GC using cBioPortal and several publicly available datasets ( Table 2). The frequencies of gene amplification were as follows: EGFR, 2.7-14.7%; ERBB2, 8-22.0%; MET, 2.2-8.8%; FGFR2, 2-5.4%. This is roughly consistent with other large cohort studies and confirms that a subset of GC harbors amplification of these RTK genes.  Tsujino et al. reported that amplification of EGFR and ERBB2 occurs in poorly differentiated adenocarcinoma and signet ring cell carcinoma, i.e., DGC [6]. The incidence of EGFR amplification is higher in metastatic tumors than that in primary tumors. Moreover, EGFR overexpression is frequently observed in GC and is associated with worse prognosis (Table 1). A large cohort study reported that ERBB2 is amplified in approximately 23% of GC cases [17]. The rate of ERBB2 amplification was substantially higher in patients with IGC than that in those with DGC in several studies that analyzed different large cohorts  (Table 1). Therefore, HER2 is less likely to play a specific role in DGC but is generally implicated in the progression of GC.
Met is a receptor for hepatocyte growth factor (HGF) and encoded by MET protooncogene [40]. Physiologically, Met plays an essential role in embryogenesis and tissue regeneration [41]. Met is aberrantly activated in various cancer types owing to gene amplification, point mutations, rearrangement, overexpression, and aberrant splicing [42]. Oncogenic activation of Met promotes diverse malignant aspects of tumors, including invasion, metastasis, drug resistance, and angiogenesis [41]. Kuniyasu et al. reported that MET amplification frequently occurs in SGC cell lines and tumor tissues [21]. Following studies also reported MET amplification in GC, although the rate differs among studies, and its association with DGC and unfavorable outcomes ( Table 1).
FGFR2 is a receptor for fibroblast growth factors, and it is oncogenically activated by genetic alterations in cancer [43]. FGFR2 is identical to the product of the K-sam gene that was cloned as a gene amplified in the KATO-III SGC cell line [44]. FGFR2 overexpression caused by gene amplification is substantially more frequent in DGC and metastatic GCs than in primary GCs, and it is associated with tumor progression and poor patient survival [43] ( Table 1).
The Cancer Genome Atlas (TCGA) project classified GC into four molecular subtypes: Epstein-Barr virus-positive, microsatellite-unstable, genomically stable, and chromosomally instable [10]. DGC is enriched in the genomically stable subtype and is characterized by recurrent E-cadherin and RhoA mutations or fusions of Rho GAPs. However, alterations in RTK genes are rare in the genomically stable subtype: EGFR (2%), ERBB2 (7%), FGFR2 (9%), and MET (0%). In contrast, a recent multiomics study demonstrated that alterations in RTK and downstream MAPK signaling pathways, mostly gene amplification of KRAS (19.4%), FGFR2 (11.2%), MET (7.1%), ERBB2 (5.1%), and EGFR (4.1%), occur more frequently in cancer cells within malignant ascites than those in primary DGC [12] (Figure 1). In addition, RTK alterations occur in a mutually exclusive manner [8,9,12]. In contrast, MET amplification has been reported to coexist with HER2 amplification in several tumor samples [18]. Interestingly, Tajiri et al. reported that HER2 was coamplified with EGFR, FGFR, and MET in some tumors, but in mutually exclusive cells [15]. Thus, the amplification of one RTK signaling component may be sufficient to drive tumor progression and can occur in a mutually exclusive manner at the cellular level. Taken together, RTK amplification is most likely a key event in the acquisition of malignant and metastatic phenotypes in DGC cells.
Loss of E-cadherin function that weakens cell-cell adhesion is a hallmark of DGC. Germline mutation of CDH1 gene encoding E-cadherin causes hereditary DGC [45]. Previous multiomics studies did not show any correlation between CDH1 mutation and RTK gene amplification [10,12]. Nevertheless, RTK signaling can negatively regulate E-cadherin function via downregulation/degradation of E-cadherin and disassembly of E-cadherin complexes [46]. Thus, RTK gene amplification and resulting oncogenic activation may contribute to the loss of E-cadherin function in DGC without CDH1 mutation.

Targeting RTKs for Peritoneal Dissemination of DGC
In general, DGC is less sensitive to cytotoxic chemotherapy than IGC [3,47]. Although the anti-HER2 antibody, trastuzumab, in combination with chemotherapy improved the survival of patients with HER2-positive advanced IGC, its efficacy was limited in patients with DGC [48]. This is most likely because the rate of HER2 amplification was low in DGC. In contrast, dozens of RTK inhibitors targeting MET, FGFR2, and EGF exhibited efficacy against DGC harboring RTK gene amplification, at least in vitro and in preclinical models (Table 3).  Tumor growth [83] (1) SMI, small molecule inhibitor; mAb, monoclonal antibody; ADC, antibody-drug conjugate; PROTAC, proteolysis targeting chimera.
Gene alterations in the FGFR family (FGFR1, FGFR2, FGFR3, and FGFR4) have been reported in various cancers. Thus, pan-FGFR inhibitors have been developed and show antitumor effects against DGC in preclinical studies, and some of them have been tested in clinical trials [43,84]. As gene amplification of FGFR2 is the predominant FGFR alteration in DGC, the development of FGFR2-specific inhibitors may improve efficacy and reduce adverse effects. In this regard, a unique selective allosteric inhibitor of FGFR2, alofanib (RPT835), was reported [85]. Although its efficacy against DGC is currently unclear, clinical studies on alofanib in patients with advanced or metastatic GC are ongoing. Other therapeutic modalities targeting FGFR2, such as a bivalent degrader, DGY-09-192 [78], a neutralizing monoclonal antibody, PRO-007 [80], and an antibody-drug conjugate, BAY 1187982 [81], may have better efficacy and selectivity than that of small-molecule inhibitors.
Although DGC cells addicted to amplified RTK signaling firstly showed high sensitivity to the RTK inhibitors, resistant cells emerge upon their continuous exposure to RTK inhibitors. As this is a serious issue in clinical usage of RTK inhibitors, the underlying molecular mechanisms have been extensively studied. Coamplification of RTKs such as HER2 and/or EGFR contribute to therapeutic resistance to Met inhibitor in DGC harboring Met amplification [86]. A patient with concurrent MET and HER2 amplification responded to combined MET/HER2 inhibition. It was reported that FGFR2 overexpression is responsible for Met inhibitor resistance in MET-amplified PDX tumors of GC [28]. In this case, treatment with FGFR2 and Met inhibitors blocked the tumor growth. Overexpression of phosphoinositide 3-kinase (PI3K) p110α contributes to acquired resistance to Met inhibitor in DGC cells with MET amplification [83]. PI-103, a PI3K inhibitor, in combination with Met inhibitor showed antitumor effects against the Met-inhibitor-resistant DGC cells. Truncated forms of RAFs were also reported to confer resistance to Met inhibition in DGC cells with MET amplification [87]. FGFR2-ACSL5 fusion was found in a patient with FGFR2-amplified GC that acquired resistance to FGFR inhibitor [88]. Acquired resistance to FGFR inhibitor also occurs via PKC-mediated GSK3β phosphorylation in DGC-derived PDX tumors with FGFR2 amplification [89]. PKC inhibitors reversed the resistance of FGFR2-addicted tumors to FGFR inhibitors in vivo. Futibatinib, a unique irreversible pan-FGFR inhibitor that binds to the FGFR kinase domain, demonstrates antitumor activity against DGC with FGFR2 amplification [70,90]. Notably, futibatinib can inhibit FGFR2 mutants resistant to ATP-competitive FGFR inhibitors [70]. Taken together, the identification of responsible and druggable molecules and the development of inhibitors with different modes of action are critical to overcoming resistance to RTK inhibitors in RTK-addicted DGC.
RTK signaling plays a pivotal tumor-supporting role not only in cancer cells, but also in the tumor microenvironment. For example, vascular endothelial growth factor receptor (VEGFR) expressed in endothelial cells promotes tumor angiogenesis. Accordingly, dualor multitarget tyrosine kinase inhibitors that inhibit both VEGFR in endothelial cells and other RTKs in cancer cells may have better therapeutic effects against RTK-addicted tumors. Several inhibitors targeting both VEGFR and Met or FGFR2 have been developed and tested in preclinical and clinical trials (Table 3). However, targeting multiple RTKs may increase the risk of adverse effects.

Novel Downstream Effectors of RTK in DGC
As described above, the use of RTK inhibitors inevitably results in acquired resistance in tumors. Therefore, understanding the downstream signaling of RTK is critical for the development of novel and alternative targeting approaches. PLEKHA5 (Pleckstrin homology domain containing A5) is a member of the PLEKHA family of proteins that contain a PH domain. Using a phosphoproteomic approach, we identified PLEKHA5 as a protein that is tyrosine-phosphorylated downstream of Met signaling (Figure 2) [91]. PLEKHA5 silencing selectively blocked the growth of DGC cells addicted to amplified Met, even when they acquired resistance to Met inhibitors. PLEKHA5 knockdown dysregulates glycolysis, leading to JNK activation and apoptotic cell death. In a mouse xenograft model, PLEKHA5 silencing markedly suppressed the peritoneal dissemination of MET-amplified DGC cells. Although its precise cellular functions remain to be elucidated, PLEKHA5 may be a biomarker for Met addiction, as well as a potential therapeutic target. patient with FGFR2-amplified GC that acquired resistance to FGFR inhibitor [88]. Acquired resistance to FGFR inhibitor also occurs via PKC-mediated GSK3β phosphorylation in DGC-derived PDX tumors with FGFR2 amplification [89]. PKC inhibitors reversed the resistance of FGFR2-addicted tumors to FGFR inhibitors in vivo. Futibatinib, a unique irreversible pan-FGFR inhibitor that binds to the FGFR kinase domain, demonstrates antitumor activity against DGC with FGFR2 amplification [70,90]. Notably, futibatinib can inhibit FGFR2 mutants resistant to ATP-competitive FGFR inhibitors [70]. Taken together, the identification of responsible and druggable molecules and the development of inhibitors with different modes of action are critical to overcoming resistance to RTK inhibitors in RTK-addicted DGC.
RTK signaling plays a pivotal tumor-supporting role not only in cancer cells, but also in the tumor microenvironment. For example, vascular endothelial growth factor receptor (VEGFR) expressed in endothelial cells promotes tumor angiogenesis. Accordingly, dualor multitarget tyrosine kinase inhibitors that inhibit both VEGFR in endothelial cells and other RTKs in cancer cells may have better therapeutic effects against RTK-addicted tumors. Several inhibitors targeting both VEGFR and Met or FGFR2 have been developed and tested in preclinical and clinical trials (Table 3). However, targeting multiple RTKs may increase the risk of adverse effects.

Novel Downstream Effectors of RTK in DGC
As described above, the use of RTK inhibitors inevitably results in acquired resistance in tumors. Therefore, understanding the downstream signaling of RTK is critical for the development of novel and alternative targeting approaches. PLEKHA5 (Pleckstrin homology domain containing A5) is a member of the PLEKHA family of proteins that contain a PH domain. Using a phosphoproteomic approach, we identified PLEKHA5 as a protein that is tyrosine-phosphorylated downstream of Met signaling ( Figure 2) [91]. PLEKHA5 silencing selectively blocked the growth of DGC cells addicted to amplified Met, even when they acquired resistance to Met inhibitors. PLEKHA5 knockdown dysregulates glycolysis, leading to JNK activation and apoptotic cell death. In a mouse xenograft model, PLEKHA5 silencing markedly suppressed the peritoneal dissemination of METamplified DGC cells. Although its precise cellular functions remain to be elucidated, PLEKHA5 may be a biomarker for Met addiction, as well as a potential therapeutic target.  Downregulation of PLEKHA5 induces apoptosis via JNK activation and blocks cell migration, invasion, and peritoneal metastasis in Met-addicted DGC cells. SHP2 is also tyrosine-phosphorylated downstream of amplified Met and FGFR. Inhibition of SHP2 blocks growth, migration, invasion, and peritoneal dissemination of Met-addicted DGC. TfR1 associates with FGFR2 and is tyrosinephosphorylated. TfR1 promotes transferrin-mediated iron uptake, which is required for growth, survival, and peritoneal metastasis of FGFR2-addicted DGC cells. SHP2 (Src homology region 2 domain-containing phosphatase 2, also known as PTPN11, protein-tyrosine phosphatase nonreceptor type 11) is an oncogenic nonreceptortype tyrosine phosphatase that regulates ERK activation downstream of RTK signaling [92]. Recurrent mutations in the PTPN11 gene encoding SHP2 have been observed in a variety of human cancers; therefore, SHP2 is thought to be a promising therapeutic target. Consequently, effective and selective allosteric SHP2 inhibitors, such as SHP099, have been developed recently [93]. We identified SHP2 by phosphoproteomic analysis of DGC cells with MET amplification [82] (Figure 2). SHP2 was tyrosine-phosphorylated in DGC cells with either MET or FGFR2 gene amplification. The growth of these cells was severely impaired by the knockdown or pharmacological inhibition of SHP2, even in Met-addicted DGC cells that acquired resistance to Met inhibitors. Furthermore, inhibition of SHP2 markedly suppressed the peritoneal dissemination of DGC cells harboring MET amplification. As SHP2 serves as a common signaling node downstream of multiple RTKs, targeting SHP2 may be an attractive alternative approach for the treatment of DGC with RTK amplification. As SHP2 is also involved in the immune checkpoint downstream of PD-1 [94], blockage of SHP2 may show additional efficacy through dual inhibition of RTK signaling and immune suppression.
Transferrin receptor 1 (TfR1) is a ubiquitously expressed membrane protein necessary for the cellular uptake of iron-loaded transferrin. TfR1 is upregulated in a variety of cancers and supports cancer cell growth by fulfilling an increased iron demand [95]. Shirakihara et al. recently identified TfR1 as a tyrosine-phosphorylated protein associated with FGFR2 in DGC harboring FGFR2 amplification [96] (Figure 2). TfR1 knockdown or FGFR2 inhibition impaired iron uptake and proliferation in DGC cells with FGFR2 amplification. Furthermore, TfR1 knockdown suppressed peritoneal metastasis of FGFR2-amplified DGC and improved survival in a mouse xenograft model. Thus, TfR1 plays a pivotal role in the oncogenic signaling of FGFR2; therefore, it may be a therapeutic target for FGFR2-addicted DGC. This finding provides a strong rationale for the clinical evaluation of TfR1 inhibitors, such as monoclonal antibodies against TfR1 [97].

Perspective
RTK amplification is a key event in the malignant progression of DGC, and it is a potential therapeutic target. However, several obstacles hinder the clinical application of RTK inhibitors in the treatment of DGC. For example, despite obvious antitumor effects in preclinical models, several clinical trials have shown insufficient benefits of RTK inhibitors in the treatment of DGC [98]. This is most likely owing to the lack of biomarkers to select patients with tumors that are addicted to RTK signaling. In addition to assessing the amount of RTK proteins or genes, evaluating the activation status of downstream effectors and/or gene expression signatures may be necessary to select RTK-addicted tumors. Detailed tumor genome profiling may enable the accurate identification of RTK-addicted tumors and patients who can benefit from RTK-targeting therapies [64].
From a biological perspective, understanding the precise oncogenic RTK functions is important for the development of effective therapies. For instance, it remains unclear whether the specificity of downstream signaling exists among the different RTKs. FGFR2 and Met may have specific and overlapping roles, which may not be shared by other RTKs, such as HER2, in tumor malignancies, according to their embryogenic and morphogenic functions. This may confer biological advantages to DGC cells by reinforcing their invasive and metastatic phenotypes. Additionally, it remains unclear why RTK activation mechanisms differ between cancer types. In the case of Met, point mutations are the major activation mechanisms in renal cancer, whereas gene amplification is predominant in DGC. Similarly, Ras gene amplification is more prevalent in DGC, whereas point mutations are dominant in lung and pancreatic cancers. These facts raise the possibility that the signaling downstream of RTK is different between activation patterns and/or is cell-context dependent. It is also critical to understand whether oncogenic RTK signaling caused by gene amplification elicits activation of specific downstream pathways. If downstream signaling differs between oncogenic and physiological RTK activation, targeting only oncogenic signaling would be an ideal therapeutic approach with minimal adverse effects. Thus, further detailed biological studies are necessary to develop effective targeted therapies against DGC harboring RTK gene amplification.