Rima M. Kulikauskas

Activated Wnt/ß-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model

This study demonstrates that in malignant melanoma, elevated levels of nuclear ß-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of ß-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/ß-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear ß-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/ß-catenin signaling.

Wnt/β-Catenin Signaling and AXIN1 Regulate Apoptosis Triggered by Inhibition of the Mutant Kinase BRAFV600E in Human Melanoma

The functional crosstalk between Wnt/β-catenin and BRAF signaling may yield more effective therapies for treating melanoma. Death to Melanoma Melanoma is a particularly aggressive form of skin cancer. Although the identification of a specific mutation in the kinase-encoding gene BRAF has facilitated the development of clinical therapies, many melanoma patients with the BRAFV600E mutation fail to exhibit long-term responsiveness to inhibition of this kinase. Biechele et al. found that some melanoma cell lines exhibited an enhanced apoptotic response when BRAFV600E was inhibited simultaneously with activation of the Wnt/β-catenin pathway. The susceptibility to this cell death response correlated with the ability of the combination treatment to reduce the abundance of AXIN1, a negative regulator of the Wnt/β-catenin pathway, and knockdown of AXIN1 abundance caused apoptosis-resistant melanoma cell lines to become susceptible to apoptosis in response to the combined treatment. Thus, exploiting this functional crosstalk between BRAF and Wnt/β-catenin signaling may yield more effective therapies for treating melanoma. Because the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAFV600E mutation, inhibits Wnt/β-catenin signaling in human melanoma cells. Because inhibitors of BRAFV600E show promise in ongoing clinical trials, we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAFV600E inhibitor PLX4720. We found that endogenous β-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/β-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of β-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAFV600E inhibitor. Thus, Wnt/β-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAFV600E, suggesting that manipulation of the Wnt/β-catenin pathway could be combined with BRAF inhibitors to treat melanoma.

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

Activation of Wnt/β-catenin signaling increases apoptosis in melanoma cells treated with trail.

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.

Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation

The limitations of revolutionary new mutation-specific inhibitors of BRAFV600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAFV600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAFV600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.

Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells

Background: Wnt/β-catenin signaling inhibits melanoma cell viability. Results: Integration of phosphoproteomics and RNA interference screens querying the Wnt/β-catenin pathway reveals protein kinase N1 as an inhibitor of signaling. Conclusion: Protein kinase N1 inhibits Wnt/β-catenin signaling and apoptosis in melanoma cells. Significance: This study identifies a kinase that inhibits Wnt/β-catenin signaling, a pathway critical to melanoma cell viability. Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

FAM123A Binds to Microtubules and Inhibits the Guanine Nucleotide Exchange Factor ARHGEF2 to Decrease Actomyosin Contractility

Unlike related proteins, FAM123A interacts with microtubule-associated proteins and alters microtubule dynamics. The Microtubule Regulator in the Family Mutations in the tumor suppressor WTX, which is also known as FAM123B, are associated with Wilms disease in children. To better define the cellular functions of the other members of the FAM123 family, Siesser et al. characterized the protein-protein interaction networks for FAM123A, WTX, and FAM123C and found that in contrast to the other FAM123 family members, FAM123A interacted with microtubule-associated proteins. Biochemical, cellular, and imaging assays indicated that FAM123A altered microtubule dynamics and limited actomyosin contractility, a property that generates the mechanical force required for changes in cell shape, which in turn can regulate cell adhesion and migration. Indeed, cells lacking FAM123A showed increased adhesion and decreased migration. The FAM123 gene family comprises three members: FAM123A, the tumor suppressor WTX (also known as FAM123B), and FAM123C. WTX is required for normal development and causally contributes to human disease, in part through its regulation of β-catenin–dependent WNT signaling. The roles of FAM123A and FAM123C in signaling, cell behavior, and human disease remain less understood. We defined and compared the protein-protein interaction networks for each member of the FAM123 family by affinity purification and mass spectrometry. Protein localization and functional studies suggest that the FAM123 family members have conserved and divergent cellular roles. In contrast to WTX and FAM123C, we found that microtubule-associated proteins were enriched in the FAM123A protein interaction network. FAM123A interacted with and tracked with the plus end of dynamic microtubules. Domain interaction experiments revealed a “SKIP” amino acid motif in FAM123A that mediated interaction with the microtubule tip tracking proteins end-binding protein 1 (EB1) and EB3—and therefore with microtubules. Cells depleted of FAM123A showed compartment-specific effects on microtubule dynamics, increased actomyosin contractility, larger focal adhesions, and decreased cell migration. These effects required binding of FAM123A to and inhibition of the guanine nucleotide exchange factor ARHGEF2, a microtubule-associated activator of RhoA. Together, these data suggest that the SKIP motif enables FAM123A, but not the other FAM123 family members, to bind to EB proteins, localize to microtubules, and coordinate microtubule dynamics and actomyosin contractility.

Abstract 4011: Wnt/β-catenin transcriptional activation promotes tumorigenesis and predicts survival in pancreatic cancer.

Mutations in key regulators of Wnt/β-catenin signaling lead to its aberrant hyperactivation and promote oncogenesis in several cancer types. While Wnt/β-catenin signaling is variably increased in pancreatic adenocarcinoma (PDAC), there is a near absence of activating mutations in its key regulatory genes in PDAC tumors. As the precise role of Wnt/β-catenin signaling in pancreatic tumorigenesis is unclear, we sought to clarify the mechanisms by which it is altered and determine its specific consequences on gene transcription and phenotype in PDAC. Using a luciferase-based Wnt/β-catenin reporter platform to determine relative levels of Wnt/β-catenin transcriptional activation, we found pathway activation was highly variable across 22 PDAC cell lines, including distinct subsets with either low or much high levels of baseline reporter activity. Supervised analysis of microarray data was performed on these defined subsets to generate a pancreatic-specific gene expression signature able to discriminate levels of Wnt/β-catenin transcriptional activation in PDAC. This analysis identified a set of 208 genes (median FDR 3.4%) representing a robust candidate list of mediators or downstream transcriptional targets. Several genes were validated as bona fide downstream transcriptional targets of Wnt/β-catenin in PDAC cell lines by observing expected changes in their expression following genetic and pharmacologic manipulations that activate or inhibit Wnt signaling. WNT7B, FZD5 and TCF7L2 were among genes commonly overexpressed in Wnt high lines, representing potential positive mediators of Wnt/β-catenin signaling in PDAC. Likewise, NLK and CSNK1E were among genes overexpressed in Wnt low lines, representing potentially important negative regulators of the pathway in PDAC. When primary patient tumors were dichotomized based on a pancreas-specific Wnt/β-catenin transcriptional signature, PDAC tumors with higher Wnt/β-catenin transcriptional activation had worse disease-specific survival (median survival time 20.3 versus 43.9 months, log rank P=0.03), suggesting a possible direct link between Wnt/β-catenin transcriptional activation and aggressive tumor behavior. Furthermore, genetic and pharmacologic inhibition of Wnt/β-catenin signaling in PDAC cell lines significantly reduced non-adherent growth and tumorsphere formation in vitro. Inhibition of Wnt/β-catenin signaling also increased survival and reduce metastatic burden in an in vivo orthotopic xenograft model using PDAC cell lines. This study has defined a pancreatic cancer-specific Wnt/β-catenin transcriptional signature able to predict more aggressive clinical behavior and with potential utility for stratifying patients most likely to benefit from Wnt-targeted therapy. Citation Format: Michael Arensman, Anna R. Lay, Rima M. Kulikauskas, Andy J. Chien, David W. Dawson. Wnt/β-catenin transcriptional activation promotes tumorigenesis and predicts survival in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4011. doi:10.1158/1538-7445.AM2013-4011

Abstract B72: Wnt7B mediates high levels of autocrine Wnt/β-catenin signaling in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/β-catenin signaling mediates diverse phenotypic outcomes in the pancreas dictated by its temporospatial context and relative levels of activation. While Wnt/β-catenin signaling is generally thought to promote tumorigenesis, it is still unclear how and to what extent the Wnt pathway is deregulated in pancreatic adenocarcinoma (PDAC) or whether its deregulation appreciably alters PDAC phenotype or patient outcome. To begin to address these important questions, Wnt/β-catenin signaling was examined in numerous PDAC cell lines by dual luciferase reporter assays and endogenous target gene expression. Two patterns of Wnt/β-catenin activity were observed across PDAC cell lines: (1) those with high constitutive Wnt/β-catenin activity and (2) those with low constitutive Wnt/β-catenin activity and variable responsiveness to Wnt ligand stimulation. Gene expression microarray analysis revealed cell lines with high constitutive Wnt activity consistently had elevated expression of WNT7B. Addressing this mechanistically, WNT7B gene knockdown studies verified Wnt/β-catenin signaling was heavily dependent upon WNT7B expression in cell lines with high levels of activity. Phenotypically, WNT7B knockdown had no appreciable effect on PDAC cell line anchorage-dependent cell growth or survival, but did significantly decreased PDAC cell line anchorage-independent cell growth and survival, as well as clonogenicity and sphere-forming capacity. Similar dual luciferase reporter and phenotypic results were obtained when cell lines were treated with IWP-2, a drug that blocks the palmitoylation and secretion of Wnt ligands. The impact of elevated Wnt/β-catenin signaling was further addressed in patient tumors by (1) examining the immunohistochemical expression of nuclear β-catenin (a widely used surrogate of Wnt/β-catenin activation) on a large tissue microarray of PDAC tumors and (2) measuring levels of an experimentally determined WNT7B gene signature on a series of gene expression arrays performed on PDAC patient tumors. In both instances, elevated Wnt/β-catenin signaling was significantly associated with improved patient survival in Kaplan-Meier and univariate analyses. This unexpected but not unprecedented finding has been shown for other tumor types such as hepatocellular carcinoma and melanoma where Wnt/β-catenin signaling also correlates with better survival. In conclusion, our findings demonstrate WNT7B and possibly other Wnt ligands are essential mediators of autocrine-mediated, Wnt/β-catenin signaling in PDAC and may be the primary determinant of differential Wnt activation seen across the spectrum of PDAC tumors. Hypothetically, Wnt ligand driven signaling may prove more amenable to Wnt-directed therapy than those instances where hyperconstitutive Wnt pathway activation is driven by genetic mutations in APC, CTNNB1 or AXIN. In this regard, future work exploring the utility of Wnt-targeted therapy in PDAC will also need to focus on the development and optimization of predictive clinical markers that are able to stratify tumors more likely to respond to such therapy. This research study received initial support from a 2008 Pancreatic Cancer Action Network-AACR Career Development Award for Pancreatic Cancer Research in memory of Seena Magowitz. This critical financial support and career development opportunities provided by the Pancreatic Cancer Action Network have been invaluable to the success of my pancreatic cancer research program and have also led to significant extramural funding from the American Cancer Society in further support of the work presented here. Citation Format: Michael D. Arensman, Anne N. Kovochich, Anna R. Lay, Rima M. Kulikauskas, Andy J. Chien, David W. Dawson. Wnt7B mediates high levels of autocrine Wnt/β-catenin signaling in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B72.