KRAS mutant non-small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Mesenchymal NSCLCs and associated “KM” cell lines are generally less responsive than their epithelial counterparts to inhibition of the RAS pathway; identifying alternative networks that promote mesenchymal NSCLC survival may lead to more effective treatments for this subtype. Non-coding microRNA (miRNA) signatures can stratify tumors into diverse molecular subtypes. By regulating numerous targets in cancer-associated signaling pathways, miRNAs can function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). Here, we derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can promote tumor suppression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by suppressing the abundance of p62, an adaptor for selective autophagy and regulator of the transcription factor NF-κB. Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator Beclin 1, the ubiquitin ligase TRAF6 and the NF-κB subunit RELA/p65. Abundance of miR-124 inversely correlated with expression of BECN1 and TRAF6 in patient NSCLC samples. These findings identify a role for miR-124 in regulating cell survival networks in a specific subtype of KRAS mutant NSCLC cell lines, which might lead to improved subtype-selective therapeutic strategies for patients.

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, co-dependencies of MEK, TAK1 and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacological studies and cell cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G2/M cell cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G2/M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NFκB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt and NFκB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NFκB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NFκB-low, Wnt-low/NFκB-high and Wnt-high/NFκB-high, designated W, N and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics.

Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date. Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3’UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression. Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3’UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124. Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells. Citation Format: Anita K. Mehta, Kevin Hua, William Whipple, Mihn-Thuy Nguyen, Rushika M. Perera, Johanns Haybaeck, Joanne Weidhass, Jeffrey Settleman, Anurag Singh. MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2017-2524

<p>KRAS and TAK1 dependency in human and mouse colon cancer.</p>

<p>Synthetic lethality of TAK1 depletion in KRAS transformed cells.</p>

<p>KRAS and TAK1 dependency in human and mouse colon cancer.</p>

<p>Computational analysis of gene expression in primary colon tumor cohorts.</p>

The KRAS protooncogene is mutated in 40 to 50% of colon cancers. In an effort to identify strategies to treat KRAS mutant colon cancers, we previously implicated the TGF-β activated kinase (TAK1) as a candidate therapeutic target that promotes the survival of KRAS dependent cancers. In follow-up studies, we have explored and investigated the detailed mechanistic basis for TAK1 mediated survival signaling in KRAS dependent cancer cells. Using proteomic and transcriptomic analyses of proinflammatory signaling mediators, we have uncovered complex autocrine/paracrine signaling loops that are constitutively activated in KRAS dependent colon cancer cells. A central mediator of these signaling loops is the BMP7-BMP receptor (BMPR1A) pathway, which functions coordinately with oncogenic KRAS to drive TAK1 and NF-κB mediated transcriptional upregulation of proinflammatory cytokines such as GM-CSF, CCL5/RANTES and IL-8. Conversely, a number of cytokines and cytokine regulators are negatively regulated by KRAS-BMPR signaling interactions, including CXCL9/MIG and IL1RN. We previously showed that TAK1 inhibition with a small molecule agent, 5Z-7-oxozeaenol promotes apoptosis in colon cancer cells. We have now determined that 5Z-7-oxozeaenol, in addition to irreversibly inhibiting TAK1 kinase activity, also transiently inhibits the MEK kinase. Therefore, combined TAK1/MEK inhibition explains the potent killing effects that we have observed in KRAS dependent colon cancer cells. To test this empirically, we have used 2 selective kinase inhibitors targeting each respective kinase, AZ-TAK1 and AZD6244 to show either single agent can induce apoptotic cell selectively in KRAS dependent cells, with TAK1 inhibition resulting in stronger killing effects. Importantly, treatment of KRAS dependent colon cancer cells with combinations of AZ-TAK1 and AZD6244 results in additive killing effects, revealing a potential therapeutic strategy for KRAS dependent cancers in the clinic. Mechanistically, MEK and TAK1 converge on the control of NF-κB and canonical Wnt-dependent transcriptional activities. Surprisingly, we find that NF-κB and Wnt signaling mutually antagonize each other in terms of cytokine expression to create a finely-tuned balance between pro-death and pro-survival signals. We hypothesize that, as a consequence, these balanced signals allow for both efficient maintenance of tumor cell survival and as well as for communication with stromal components in the tumor microenvironment. Thus, KRAS, MEK or TAK1 blockade results in a tipping of the balance to favor pro-death signals. Citation Format: Kelsey L. McNew, William J. Whipple, Anita K. Mehta, Trevor Grant, Anurag Singh. MEK and TAK1 signaling interactions coordinately regulate inflammation and apoptosis in KRAS dependent colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2015-3188