Mitogen-activated protein kinase signaling causes malignant melanoma cells to differentially alter extracellular matrix biosynthesis to promote cell survival

Molecular studies have determined that aberrant signaling in the mitogen-activated protein kinase (MAPK) signaling cascade (Raf-MEK1/2-ERK1/2) is a crucial step in melanocytic neoplasia, ultimately deregulating the activity of key transcription factors that control cell growth, differentiation, and survival [1]. Specifically, gain of function mutations in which the 600 valine residue of the BRAF kinase is substituted with glutamic acid (BRAF^V600E) are present in ~40 % of patients presenting with malignant melanoma [2]. Survival is enhanced if detection, treatment and surgical excision occur when the tumor burden is restricted to a primary site; however, once the disease has metastasized to distant organs such as the lung, brain, bone, and liver, prognosis is poor, with a median survival of ~ 9 months [3]. These dismal statistics are in part due to the intrinsic resistance of melanoma to platinum-based chemotherapeutics and its rapid acquisition of resistance to initial therapeutic interventions [4, 5]. Present treatment strategies involve small molecule inhibitors of this mutant BRAF kinase, such as vemurafenib and dabrafenib [6, 7]. However, these therapies are only effective in a subset of patients: those that harbor the more prevalent BRAF^V600E and the less common BRAF^V600K mutations [8]. While drugs targeting this specific isoform are among the most promising melanoma treatments, patients eventually relapse due to reactivation of the MAPK signaling cascade, specifically ERK activity [5, 9]. To overcome de novo resistance, a combinatorial treatment of vemurafenib with a MEK inhibitor is administered in an effort to combat reactivation of the MAPK pathway [10‐12]. However, mechanisms that underlie acquired resistance after treatment with multiple inhibitors of this pathway remain elusive.

Drug resistance has been shown to be mediated by tissue architecture and cell-adhesion [13, 14]. In particular, cell adhesion-mediated drug resistance (CAM-DR) is an emergent phenotype associated with cell-cell adhesion or 2D adhesion to extracellular matrices. Myeloma cells cultured as monolayers that had adhered to fibronectin were resistant via upregulation of α4 β1 integrin compared to cells treated in suspension [15]. Similarly, tumor cells grown as spheroids show increased resistance to therapy compared to the same cells that are dissociated and grown as monolayers [16]. However, the observed acquired drug resistance following multiple targeting of the MAPK pathway is not readily explained by CAM-DR [12]. Because this reactivation attenuates drug response, it may also contribute to the development of acquired resistance [12].

The tumor microenvironment is emerging as a critical factor in malignant progression, metastasis and tumor etiology [17, 18]. To explore mechanisms that drive tumors to overcome and survive under unfavorable conditions, we aimed to delineate tumor-induced microenvironmental responses to the stress induced by drug therapeutics. Tumor cells actively modulate the host environment by secreting cytokines that reprogram stromal cells to change the extracellular matrix (ECM) milieu, thus creating a de novo microenvironment [17, 18]. While immunotherapy and monoclonal antibodies targeting tumor angiogenesis have shown promising results, many microenvironmental targets remain underexplored [18]. For example, overexpression of secreted ECM proteins such as fibronectin (FN) has been found in several solid carcinomas, and postulated to be beneficial for tumor growth and instrumental in the establishment of an ideal microenvironment [19]. Furthermore, heterogeneous expression of ECM components within tumors has been observed [20]. Pathologists have long associated the presence of abundant ECM proteins in tumors with poor prognosis and an expected dismal response to therapeutic intervention [21].

Recently, a study showed that non-small cell lung cancer cells induced FN biogenesis via p38 MAPK in response to treatment with cetuximab (targeting the EGF receptor upstream of the MAPK signaling pathway) [22]. This response was found to blunt the cytotoxic effects of cetuximab and reduced sensitivity to radiotherapy in in vitro and in vivo murine models. FN biogenesis may also reduce the efficacy of drugs targeting the BRAF kinase. Earlier observations found that a cocoon of ECM proteins, including FN, laminin, collagen IV and Tenascin C, protect small lung cancer cells from chemotherapy-induced apoptosis [23]. We hypothesized that melanoma cells modulate secretion of not only FN, but also other ECM molecules to survive drug treatment. An important question is whether baseline ECM expression per se can predict cell survival and drug resistance. Furthermore, is upregulation of ECM proteins a reaction to evolutionary pressure following drug treatment, the result of selection for pre-existing resistant subpopulations, or a combination of both? Seeking to identify and determine the temporal regulation of the secreted ECM proteins, we focused on two isogenic cell lines to mimic intratumoral heterogeneity. Our results indicate that tumor cells adjust their 3D microenvironment by modulating secretion of FN and Tenascin-C (TNC), thereby blunting the effects of MAPK pathway inhibition. We show that only clones that can modulate their ECM secretion in response to pharmaceutical stress survive. Mechanistically, FN biogenesis via p38 MAP kinase via β1 integrin is induced following pharmaceutical treatment of the MAPK signaling pathway. Also, our data suggests that biogenesis of TNC is an possible alternative to impaired FN production. This study indicates a critical role for de novo ECM biogenesis as a mechanism driving acquired resistance to drugs targeting MAPK signaling in cutaneous melanoma.

Human melanoma cell lines, A375 (cat. No. CRL-1619) and A375.S2 (cat. No. CRL-1872) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured on tissue culture flasks as described by the ATCC. A375 and A375.S2 cells were grown as monolayers in DMEM high glucose media (Life Technologies, Carlsbad, CA) supplemented with final volume of 10 % Fetal Bovine Serum (FBS), 1 % MEM non-essential amino acids, L-Glutamine, Penicillin and Streptomycin. Cells cultured with the media was refreshed every 2-3 days.

Treatment strategies for metastatic malignant melanoma have significantly improved due to our current understanding of the molecular basis of tumor etiology [29]. Targeted therapy based on BRAF V600E status has yielded dramatic results and disease regression has been observed. In spite of recent progress, the stark reality is that current treatments do not yet achieve durable responses due to melanoma intratumoral heterogeneity. The remodeling of the ECM results in changes within the microenvironment adding further complexity to the determinants of drug efficacy and the likelihood of acquired resistance. We present a novel mechanism by which cells tune their ECM secretion, namely FN via p38 MAPK –β1 integrin, in response to pharmaceutical inhibition of the MAPK signaling pathway in cutaneous melanoma cell lines, especially in 3D culture system. Here we show that when FN biogenesis is impaired, increased TNC expression is observed, suggesting a compensatory mechanism to attenuate drug treatment. By using the A375 cell lines that mimic tumor heterogeneity, we determined that only clones that can modulate FN biogenesis survive pharmaceutical intervention. These results, in conjunction with similar observations in patient data, suggest a role for ECM biosynthesis in the emergence of de novo resistance.

Extracellular matrix proteins (ECMs) are often observed to be abundantly expressed by tumor cells in comparison to their normal counterparts. Analysis of patient data confirmed that overexpression of ECM proteins such as FN and TNC are likewise observed in patients that had acquired de novo resistance from initial drug therapies. Similar outcomes have been noted for breast, laryngeal, urothelial and non-small-cell lung cancers [22, 30‐32]. Using proteomics and immunofluorescence, we determined a distinct temporal distribution of secreted ECM components and visualized the assembled ECM scaffold for two isogenic malignant melanoma clones. We determined that melanoma cells modulate the secretion of many ECM proteins that are also implicated as potential biomarkers for patients suffering from metastatic disease [33]. Recently, ECM signatures were assigned to breast and ovarian tumors of differing metastatic potential [34, 35]. These ECM signatures were not solely derived from the stromal cell secretions, but also included contributions from the tumor cells themselves [35]. Here, we go a step further to follow the evolution of a melanoma-derived ECM signature. In particular, the secretion patterns of the two clones were significantly different with respect to relative amounts of FN, periostin, TNC, laminins and vitronectin (Fig. 1c). FN also differed architecturally; fibrils were predominantly observed in one clone vs. globular scaffolds for the other (Fig. 2d). FN fibril assembly is regulated by tensional reorganization of actin via stress fibers for cells cultured in 2D [36‐38]. Actin structures convey different mechanical properties based on spatial organization in 2D, and regulate specific motilities in 3D [25, 39]. Furthermore, cell motility regulates ECM remodeling and plays a role in the final architecture of the ECM scaffold [40]. These two cell lines show differential adhesion forces when placed in a 2D biomimetic platform simulating cell-ECM interactions [41]. Additionally, differences in FN conformation dictate the availability of binding sites that affect signaling cascades associated with growth, migration and adhesion [42, 43]. In our system, it remains to be seen if there is a difference in tensional regulation and whether globular vs. fibril conformations show differential drug sensitivity.

In this study, we observed that FN was differentially secreted and assembled when tumor cells harboring the BRAF^V600E mutation were cultured in 2D vs. in a biomimetic 3D platform [44]. In vivo, tumor cells have access to various sources of fibronectin, namely the plasma and stroma. As a surrogate basement membrane gel, the laminin-rich ECM contains several ECM proteins and cytokines, including minute amounts of FN [40], However, previous studies have shown that FN-null cells poorly assemble FN fibrils when exogenous FN is added to them [45]. We confirmed that a reduced number of shFN cells assembled exogenous FN compared to Scr cells in in vivo and in vitro 3D culture (Figs. 3 and 7). Growth inhibition when FN has been compromised has also been observed for other types of cancer [46]. This was due in part to reduced expression of β1 integrin and alterations of the cell cycle; however, in our system the short hairpin did not alter the cell cycle or reduce β1 integrin levels. Melanoma-derived conditioned media was shown to stimulate in vivo FN expression in target organs preceding future metastatic lesions. This phenomenon was also attributed to resident fibroblasts that create the “pre-metastatic niche” [47]. Our data suggests that metastatic melanoma also modulates its “tumor niche” by secreting and assembling its own ECM scaffold to survive drug treatment. Our results suggest that modulation of FN secretion serves as a response to external stress and may be separate from the remodeling of stromal components observed during initial malignant transformation.

Melanoma is largely resistant to platinum treatments since several factors such as drug uptake, increased efflux, extracellular interaction via cell adhesions and slow cycling cells may contribute to intrinsic resistance [48‐50]. In our system 3D architecture did mitigate drug efficacy, as only a partial killing was achieved for FN-null tumor aggregates and minimal FN secretion was observed in response to cisplatin.

It has been previously reported that activation of the ERK/MAP kinase in melanoma cells stimulates FN biogenesis via the early growth response-1 transcription factor (Egr-1) [51]. High FN and Egr-1 levels were found in BRAF^V600E mutant cells and patient tumors. However, stimulation of the ERK pathway was insufficient to induce FN biogenesis in normal melanocytes [51]. Here, inhibition of the ERK pathway activates FN biogenesis via p38MAPK and β1 integrin signaling in melanoma cells but not in melanocytes. In a previous study, β1 integrin inhibition targeting FN has increased radio sensitization of breast tumor cells in 3D culture [52]. However, non-malignant breast cells undergo apoptosis following treatment with a β1-integrin function-blocking antibody, whereas the malignant counterpart cells appear resistant to this treatment [53, 54]. Integrin blocking treatment is successful only if there is a differential response between normal tissue and its malignant counterpart. Thus, in addition to multiple targets of MAPK signaling, concomitant treatment with a p38 inhibitor may be a better strategy to prevent acquire drug resistance due to FN biosynthesis. However, in the case of metastatic disease, acute treatment with integrin-blocking antibodies may be useful to overcome de novo resistance. Nevertheless, alternative strategies to overcome FN mediated cell adhesion mediated drug resistance have been reported that could reverse drug resistance in leukemia and solid tumors [55, 56].

In normal cells during early embryogenesis, FN provides a scaffold for the assembly of other ECM proteins to direct organ specificity and fate, so we reasoned that other ECM proteins would be altered when FN biogenesis is affected [57]. In fact, we observed an increase in Tenascin-C biogenesis by shFN melanoma cells. Here, we show that drug treatment may induce ECM biosynthesis, contributing to de novo resistance. This must be considered when designing therapeutic interventions. Our findings suggest that modulation of tumor ECM biogenesis mimics the behavior of bacteria that assemble a biofilm in response to nutrient deficiencies, antibiotics and oxidative stress [58]. This biofilm acts as a physical barrier and signaling reservoir to communicate sensing of deleterious conditions. Tumor cells may use this secreted “biofilm” to communicate and communally enter a state of dormancy until environmental conditions are more favorable. If we can understand the mechanisms underlying tumor biofilm assembly, we may be able to seize a novel opportunity for therapeutic intervention.