SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

Up-regulation of N-cadherin by Collagen I-activated Discoidin Domain Receptor 1 in Pancreatic Cancer Requires the Adaptor Molecule Shc1.

Pancreatic ductal adenocarcinomas are highly malignant cancers characterized by extensive invasion into surrounding tissues, metastasis to distant organs, and a limited response to therapy. A main feature of pancreatic ductal adenocarcinomas is desmoplasia, which leads to extensive deposition of collagen I. We have demonstrated that collagen I can induce epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. A hallmark of EMT is an increase in the expression of the mesenchymal cadherin N-cadherin. Previously we showed up-regulation of N-cadherin promotes tumor cell invasion and that collagen I-induced EMT is mediated by two collagen receptors, α2ß1-integrin and discoidin domain receptor 1 (DDR1). DDR1 is a receptor-tyrosine kinase widely expressed during embryonic development and in many adult tissues and is also highly expressed in many different cancers. In the signaling pathway initiated by collagen, we have shown proline-rich tyrosine kinase 2 (Pyk2) is downstream of DDR1. In this study we found isoform b of DDR1 is responsible for collagen I-induced up-regulation of N-cadherin and tyrosine 513 of DDR1b is necessary. Knocking down Shc1, which binds to tyrosine 513 of DDR1b via its PTB (phosphotyrosine binding) domain, eliminates the up-regulation of N-cadherin. The signaling does not require a functional SH2 domain or the tyrosine residues commonly phosphorylated in Shc1 but is mediated by the interaction between a short segment of the central domain of Shc1 and the proline-rich region of Pyk2. Taken together, these data illustrate DDR1b, but not DDR1a, mediates collagen I-induced N-cadherin up-regulation, and Shc1 is involved in this process by coupling to both DDR1 and Pyk2.

Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover.

Desmosomes are prominent adhesive junctions present between many epithelial cells as well as cardiomyocytes. The mechanisms controlling desmosome assembly and remodeling in epithelial and cardiac tissue are poorly understood. We recently identified protein palmitoylation as a mechanism regulating desmosome dynamics. In this study, we have focused on the palmitoylation of the desmosomal cadherin desmoglein-2 (Dsg2) and characterized the role that palmitoylation of Dsg2 plays in its localization and stability in cultured cells. We identified two cysteine residues in the juxtamembrane (intracellular anchor) domain of Dsg2 that, when mutated, eliminate its palmitoylation. These cysteine residues are conserved in all four desmoglein family members. Although mutant Dsg2 localizes to endogenous desmosomes, there is a significant delay in its incorporation into junctions, and the mutant is also present in a cytoplasmic pool. Triton X-100 solubility assays demonstrate that mutant Dsg2 is more soluble than wild-type protein. Interestingly, trafficking of the mutant Dsg2 to the cell surface was delayed, and a pool of the non-palmitoylated Dsg2 co-localized with lysosomal markers. Taken together, these data suggest that palmitoylation of Dsg2 regulates protein transport to the plasma membrane. Modulation of the palmitoylation status of desmosomal cadherins can affect desmosome dynamics.

Palmitoylation of plakophilin is required for desmosome assembly.

Desmosomes are prominent adhesive junctions found in various epithelial tissues. The cytoplasmic domains of desmosomal cadherins interact with a host of desmosomal plaque proteins, including plakophilins, plakoglobin and desmoplakin, which, in turn, recruit the intermediate filament cytoskeleton to sites of cell-cell contact. Although the individual components of the desmosome are known, mechanisms regulating the assembly of this junction are poorly understood. Protein palmitoylation is a posttranslational lipid modification that plays an important role in protein trafficking and function. Here, we demonstrate that multiple desmosomal components are palmitoylated in vivo. Pharmacologic inhibition of palmitoylation disrupts desmosome assembly at cell-cell borders. We mapped the site of plakophilin palmitoylation to a conserved cysteine residue present in the armadillo repeat domain. Mutation of this single cysteine residue prevents palmitoylation, disrupts plakophilin incorporation into the desmosomal plaque and prevents plakophilin-dependent desmosome assembly. Finally, plakophilin mutants unable to become palmitoylated act in a dominant-negative manner to disrupt proper localization of endogenous desmosome components and decrease desmosomal adhesion. Taken together, these data demonstrate that palmitoylation of desmosomal components is important for desmosome assembly and adhesion.

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance.

Small-cell lung cancer (SCLC) is designated a recalcitrant cancer due to its five-year relative survival rate of less than 7%. First line SCLC treatment has changed modestly in the last 40 years. The NeuroD1 subtype of SCLC (SCLC-N) commonly harbors MYC amplifications and other hallmarks of aggressive behavior. Finding novel therapeutic options that effectively eliminate residual disease observed after initial response to therapy is essential to improving SCLC patient outcome. Here we show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade is critical for clonogenicity and tumor initiation in vitro and in vivo in the highly aggressive, metastatic and therapy resistant NeuroD1 subtype of SCLC. Tumor-initiating cells (TICs) are reported as the sanctuary population within the bulk tumor responsible for therapeutic resistance and relapse. Previous studies concluded ERK activation was inhibitory to growth and tumor development. We show that signaling through KSR1 is conserved in SCLC-N and that it regulates tumor initiation through interaction with ERK. We further show that KSR1 mediates cisplatin resistance in SCLC-N cells. While 50% of control SCLC-N cells show resistance after 6 weeks of exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC-N cells. KSR1 KO also significantly enhances the ability of cisplatin to decrease SCLC-N TICs, indicating that targeting KSR1 might be selectively toxic to cells responsible for therapeutic resistance and tumor initiation. Thus, KSR1 function in SCLC-N serves as a novel model for understanding the role of KSR1-dependent signaling in normal and malignant tissues. These findings shed light on a key distinct protein responsible for regulation in SCLC-N tumors, and a potential subtype specific therapeutic target.

PGC-1ß and ERRα Promote Glutamine Metabolism and Colorectal Cancer Survival via Transcriptional Upregulation of PCK2.

BACKGROUND: Previous studies have shown that Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1ß) and Estrogen-Related Receptor Alpha (ERRα) are over-expressed in colorectal cancer and promote tumor survival. METHODS: In this study, we use immunoprecipitation of epitope tagged endogenous PGC-1ß and inducible PGC-1ß mutants to show that amino acid motif LRELL on PGC-1ß is responsible for the physical interaction with ERRα and promotes ERRα mRNA and protein expression. We use RNAsequencing to determine the genes regulated by both PGC-1ß & ERRα and find that mitochondrial Phosphoenolpyruvate Carboxykinase 2 (PCK2) is the gene that decreased most significantly after depletion of both genes. RESULTS: Depletion of PCK2 in colorectal cancer cells was sufficient to reduce anchorage-independent growth and inhibit glutamine utilization by the TCA cycle. Lastly, shRNA-mediated depletion of ERRα decreased anchorage-independent growth and glutamine metabolism, which could not be rescued by plasmid derived expression of PCK2. DISCUSSION: These findings suggest that transcriptional control of PCK2 is one mechanism used by PGC-1ß and ERRα to promote glutamine metabolism and colorectal cancer cell survival.

KSR1- and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here, we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

The majority of cancer deaths result from tumor cells spreading to other parts of the body via a process known as metastasis. 90% of all cancers originate in epithelial cells that line the inner and outer surface of organs in our bodies. Epithelial cells, however, are typically stationary and must undergo various chemical and physical changes to transform in to migratory cells that can invade other tissues. This transformation process alters the amount of protein cells use to interact with one another. For example, epithelial cells from the colon produce less of a protein called E-cadherin as they transition into migrating cancer cells and make another protein called N-cadherin instead. A protein called KSR1 is a key component of a signaling pathway that is responsible for generating the proteins colon cancer cells need to survive. But it is unknown which proteins KSR1 helps synthesize and whether it plays a role in the metastasis of colon cancer cells. To investigate this, Rao et al. studied the proteins generated by cancerous colon cells cultured in the laboratory, in the presence and absence of KSR1. The experiment showed that KSR1 increases the levels of a protein called EPSTI1, which colon cancer cells need to transform into migratory cells. Depleting KSR1 caused cancer cells to generate less EPSTI1 and to share more features with healthy cells, such as higher levels of E-cadherin on their surface and reduced mobility. Adding EPSTI1 to the cancer cells that lacked KSR1 restored the traits associated with metastasis, such as high levels of N-cadherin, and allowed the cells to move more easily. These findings suggest that KSR1 and EPSTI1 could be new drug targets for reducing, or potentially reversing, the invasive behavior of colon cancer cells. However, further investigation is needed to reveal how EPSTI1 is generated and how this protein helps colon cancer cells move and invade other tissues.

Expression artifact with retroviral vectors based on pBMN.

While characterizing various splice forms of p120 catenin, we observed what appeared to be a novel posttranslational modification of p120, resulting in a higher molecular weight form that was dependent on the splicing pattern. Further investigation revealed the higher molecular weight form to be a fusion protein between sequences encoded by the retroviral vector and p120. We found that the publicly available sequence of the vector we used does not agree with the experimental sequence. We caution other investigators to be aware of this potential artifact.

Modulating the strength of cadherin adhesion: evidence for a novel adhesion complex.

Adherens junctions and desmosomes are critical for embryogenesis and the integrity of adult tissues. To form these junctions, classical cadherins interact via alpha- and beta-catenin with the actin cytoskeleton, whereas desmosomal cadherins interact with the intermediate filament system. Here, we used a hormone-activated mutant N-cadherin expressed in fibroblasts to show the existence of a novel classical cadherin adhesion system. N-cadherin was fused at its C-terminus to a modified estrogen receptor ligand-binding domain (NcadER) that binds 4-hydroxytamoxifen (4OHT) and expressed in L cells, which lack an endogenous cadherin. Cells with the mutant cadherin (LNER cells) aggregated in the absence of 4OHT, but only in its presence formed tightly compacted aggregates like those formed by L cells expressing wild-type N-cadherin (LN cells). Compaction of LNER cells treated with 4OHT was accompanied by elevated levels of p120ctn in NcadER immunoprecipitates, compared to immunoprecipitates of non-treated cells, but without changes in alpha- and beta-catenin, or actin. Compaction induced by 4OHT was also accompanied by increased interaction of the NcadER with the cytoskeleton and increased vimentin organization. Vimentin co-immunoprecipitated with the NcadER/catenin complex, suggesting an interaction between cadherin and vimentin. The mechanism by which vimentin interacts with the cadherin appears to involve p120ctn as it co-immunoprecipitates and colocalizes with vimentin in the parent L cells, which lack a cadherin and alpha- and beta-catenins. Disrupting the actin cytoskeleton with cytochalasin B inhibited aggregation, whereas knocking down vimentin with specific siRNAs inhibited compaction. Based on our results we propose that a vimentin-based classical cadherin complex functions together with the actin-based complex to promote strong cell-cell adhesion in fibroblasts.