Therapeutic Targets for Skin Cancer: MET Signaling and RAS Converge on EGFR

New findings from Science Signaling (21 Jun 2016: Vol. 9, Issue 433, pp. ra62 DOI: 10.1126/scisignal.aaf5106), this article indicates that EGFR pathway may offer potential therapeutic targets for patients. Here we provide part of this wonderful article. Read full details: http://stke.sciencemag.org/content/9/433/ra62.

Introduction

Studies using the multistage induction of squamous cancers on mouse skin have revealed much about the biology of tumor formation and first defined the operational and functional distinctions of initiation, promotion, premalignant progression, and malignant conversion. Furthermore, the standard protocol of 7,12-dimethylbenz[a]anthracene (DMBA) followed by 12-O-tetradecanoylphorbol 13-acetate (TPA) application confirmed the primacy of Hras mutations as an initiating event for squamous tumors in vivo and displayed the importance of regenerative hyperplasia and inflammation as the selective forces in tumor promotion, leading to the emergence of HRAS-initiated tumors. Through genetic modification of mice, the identification of potential initiating events for skin tumors has expanded to include Kras and Nras as well as known RAS targets in the epidermal growth factor receptor (EGFR) pathway, such as EGFR, ErbB2, or SOS, and more downstream factors such as v-FOS, c-MYC, IGF1, and components of the nuclear factor κB (NF-κB) pathway. In most of these cases, a proinflammatory tumor promoter is also required for tumor formation. This requirement stimulated research to elucidate how promoter-induced inflammation provides the selection stimulus for tumor outgrowth and led to a broader understanding of the role of inflammatory cells, chemokines, and molecular pathways in carcinogenesis in that they have both protective and negative consequences.

It is now recognized that initiating events themselves can have consequences on the inflammatory milieu, and this can be essential to their oncogenic potential. For example, transduction of keratinocytes with oncogenic Hras activates EGFR signaling, leading to release of interleukin-1α (IL-1α), activation of NF-κB, and elaboration of CXC motif chemokine receptor 2 (CXCR2) ligands that are essential components for HRAS-mediated keratinocyte neoplastic transformation. The magnitude of induction of these intermediate pathways is greatly enhanced by activation of protein kinase Cα (PKCα), yet the overexpression and activation of PKCα in mouse skin are not sufficient to initiate tumors in the absence of Ras mutations. Nevertheless, transgenic mice that overexpress PKCα in the epidermis (K5-PKCα mice) are exquisitely sensitive to tumor promotion after DMBA initiation. This exquisite sensitivity to tumor promotion provides a model that could help identify initiating events of less potency than Ras mutations but of great relevance to human cancer. Signaling by hepatocyte growth factor (HGF) through its receptor tyrosine kinase MET has been studied in multiple epithelial carcinomas. Several studies suggest that MET protein is abundant in a subset of human skin cancers and various other epithelial cancers. In human cancers where MET appears to contribute, the abundance of MET protein is most frequently increased as a result of transcriptional up-regulation or gene amplification. Constitutively active MET mutations have been identified in hereditary papillary renal cell carcinoma patients, but in general, activating MET mutations are infrequent. The downstream targets of activated MET include the adapter protein GAB1, the phospholipase PLC-γ, the guanosine triphosphatase RAS, and the kinases PI3K, RAF, ERK, and MAPK, leading to mitogenic, motogenic, and morphogenic responses in many cell types. Aside from intrinsic changes in MET, mutations in the regulatory region of the gene encoding HGF that induce its overexpression and activation of MET contribute to both breast and bladder cancer. Copy number amplification of HGF has been identified in head and neck squamous cell carcinoma (HNSCC), and HGF is described as a cancer driver gene across squamous cancers independent of tissue origin. HGF mutations have also been detected in about 20% of human cutaneous SCCs, and HGF expression increases as a primary response to ultraviolet (UV) light exposure in the skin. In most normal tissues, HGF is produced predominantly by stromal cells in the microenvironment and elicits both paracrine and autocrine MET signaling. Thus, an experimental model that links HGF and MET to squamous cancer has relevance for various human cancers. In transgenic mice overexpressing HGF driven by the metallothionein (MT) 1 promoter (MT-HGF mice), melanocytes relocate to the epidermal-dermal junction, closely approximating the cellular distribution of human skin. A single dose of UV irradiation or DMBA followed by TPA in MT-HGF neonates induces melanomas and SCCs through the activation of MET. Here, we used the MT-HGF mouse in combination with the tumor promotion–sensitive K5-PKCα mouse to explore the stage-specific contribution of increased MET activity to the development of squamous cell tumors in the skin. In this setting, we found that HGF-activated MET was a fully functional tumor initiator for skin tumor formation, and the downstream signaling from MET activation co-opted many of the properties of oncogenic RAS, signaling in keratinocytes indicating that together HGF and MET comprise a relevant oncogenic pair for the propagation of human skin cancers.

Discussion

Our work addresses the early changes required for the conversion of a normal keratinocyte into a premalignant tumor on the road to forming a cancer. The discoveries in the mid-1980s that an activated Hras allele is present in nearly all benign tumors induced on mouse skin by DMBA-TPA exposure together with experimental evidence that activated HRAS is sufficient to initiate normal keratinocytes to produce benign tumors were milestones in carcinogenesis research. In the interim decades, numerous laboratories have filled in the intricate cascading signaling molecules downstream from oncogenic Ras alleles in multiple model systems. In mouse keratinocytes, crucial biochemistry for oncogenic HRAS initiation appears to involve the up-regulation of ligands for and autocrine activation of EGFR. Because RAS is downstream from EGFR signaling, this requirement for EGFR activation may indicate that the signal strength of a single mutated Rasallele is not sufficient to drive early neoplasia. After EGFR activation, IL-1α is released and activates IL-1R on keratinocytes, creating a second autocrine loop, leading to the activation of NF-κB signaling that modifies expression of specific keratinocyte genes involved in tumor formation including increasing expression and release of CXC ligands such as CXCL1. The consequences of cytokine release are both autocrine, stimulating tumor cell migration through activation of keratinocyte CXCR2, and paracrine, attracting immune cells into the tumor stroma (oncogene-induced inflammation). Inhibition of any one of these autocrine loops impairs tumor formation. We now show that activation of keratinocyte MET through elevated autocrine or paracrine HGF in the skin microenvironment uses these same pathways to be oncogenic for keratinocytes and is sufficient for tumor formation in the absence of Ras mutations but in the presence of a strong promoting stimulus such as wounding or enhanced cutaneous PKCα. The focal nature of tumor formation in the HGF-MET mice suggests that a subpopulation of keratinocytes in the skin epithelium is particularly sensitive to MET activation and the promoting stimulus. In normal mouse skin, HGF is detected in the hair follicle dermal papilla and MET in the hair follicle including the hair follicle bulge where stem cells reside. In the MT-HGF mouse, the expression of HGF is more widespread. Therefore, it is reasonable to speculate that bulge stem cells give rise to MET-induced squamous tumors, but further studies are required to support this possibility. The combination of DMBA and MT-HGF favors the selection of Kras mutant tumors in addition to the expected Hras mutant tumors, potentially arising from the same cell compartment. The skin carcinogenesis literature is peppered with examples of Kras mutant tumors emerging after carcinogen initiation when the promoting environment is modified from the standard TPA protocol. In our model, it is notable that only mutant Hras tumors and no mutant Krastumors were detected after DMBA in the DT group, emphasizing the importance of context in the selection of incipient mutant tumor cells. Furthermore, many of the tumors in this group were at lower risk for malignant conversion, whereas the increased frequency of Kras mutations in the DMBA-MT-HGF group might have contributed to malignant conversion.

The mechanism through which cell-autonomous or paracrine activation of MET in keratinocytes produced tumors became clearer when we determined that, phenotypically and biochemically, these keratinocytes reproduced the biology of RAS-transformed keratinocytes. Like RAS, MET activates EGFR through enhancing expression of the EGFR cognate ligands and controlling their maturation through the membrane-bound protease ADAM17, thus establishing the autocrine loops necessary for tumor formation. Although previous studies have shown that EGFR can lead to MET activation, our data indicate that EGFR is an obligatory effector of MET-driven mouse skin carcinogenesis. Blocking EGFR reverses the MET biochemical signature and causes MET-driven tumors to regress. Pharmacological inhibition of EGFR causes major changes in the gene expression profile of MT-HGF keratinocytes. This reliance on EGFR activity for tumor growth in vivo is also true for oncogenic RAS, further highlighting the commonalities among those two initiators of skin carcinogenesis. The crosstalk between MET and EGFR in our model is unidirectional because oncogenic RAS (and subsequent EGFR activation) does not cause MET activation, and treatment of DT keratinocytes with an EGFR inhibitor does not decrease phosphorylated MET levels.

Beyond the establishment of the autocrine loops emanating from EGFR activation, the pathways involved in both MET and RAS initiation of keratinocyte neoplasia converged on the expression of many genes and common pathways. The vast majority of the aberrantly expressed genes from keratinocytes initiated by activated RAS or MET overlapped and were concordant. Selecting the 372 most modulated and concordant RAS and MET genes to produce a highly enriched tumor-associated gene expression data set coupled with GO analyses derived from GSEA allowed us to identify biologically meaningful and coherent sets of gene functions involved. The concordant gene list confirmed the changes in cytokines, growth factors, and differentiation markers that we have come to know as the signature of initiation. Not surprisingly, the functional gene set analysis confirmed the importance of pathways associated with epidermal development and keratinocyte differentiation but revealed two unexpected highly relevant pathways. A strong functional association with endopeptidase-peptidase activity was revealed. Matrix metalloproteinases (MMPs) have been associated with transformation of keratinocytes and a number of other cancers, but the profiling revealed an association with a number of type II transmembrane serine proteases. The functions of members of this family are not well known, but TMPRSS13 is reported to activate pro-HGF to the mature ligand and TMPRSS11E is reported to decrease in HNSCC. This is an area worthy of further study. Pathways controlling lipid biosynthesis, lipid transport, and fatty acid synthesis were also unexpectedly revealed in the functional enrichment analysis. Recent studies indicate a strong association among EGFR signaling, lipid metabolism, and cancer growth. Much of the data are derived from glioblastomas, but our results suggest that the concept may be more widespread.

It is premature to speculate on what regulates the expression of downstream effectors in the RAS/MET signature profiles, but the IPA Upstream Regulator tool identified several transcriptional regulators previously associated with skin carcinogenesis or RAS transformation. Among these, reduction in p53-regulated genes has been associated with skin tumor progression and NUPR1 is required for RAS transformation of pancreatic cells. It is notable that HRAS, KRAS, and RAF1 all appear on the upstream regulator algorithm as does tumor necrosis factor (TNF) representing the key role of NF-κB in the transformation process.

Although our study focused on the role of HGF-MET in cutaneous cancer, MET signaling has been linked to a broader variety of human cancers, prompting the development of MET inhibitors as cancer therapeutics. Both preclinical and clinical experiences have revealed crosstalk of MET and EGFR in the therapeutic setting. In particular, MET amplification is identified as a resistance mechanism for tumor cell lines and lung cancer patients treated with EGFR inhibitors. Conversely, EGFR inhibitors enhance the antitumor activity of MET inhibitors in cell lines, xenograft models, and lung cancer patients. Thus, clinical trials of combined anti-MET and anti-EGFR treatment in advanced internal cancers show promise. Our data suggest that cutaneous cancers, perhaps in the setting of organ transplant patients where cutaneous SCC can be life-threatening, should be considered as a model for combined MET-EGFR kinase inhibitor therapy where the dynamics of tumor response can be followed visually and sampled temporally.