Verline Justilien

The PRKCI and SOX2 Oncogenes Are Coamplified and Cooperate to Activate Hedgehog Signaling in Lung Squamous Cell Carcinoma

We report that two oncogenes coamplified on chromosome 3q26, PRKCI and SOX2, cooperate to drive a stem-like phenotype in lung squamous cell carcinoma (LSCC). Protein kinase Cι (PKCι) phosphorylates SOX2, a master transcriptional regulator of stemness, and recruits it to the promoter of Hedgehog (Hh) acyltransferase (HHAT) that catalyzes the rate-limiting step in Hh ligand production. PKCι-mediated SOX2 phosphorylation is required for HHAT promoter occupancy, HHAT expression, and maintenance of a stem-like phenotype. Primary LSCC tumors coordinately overexpress PKCι, SOX2, and HHAT and require PKCι-SOX2-HHAT signaling to maintain a stem-like phenotype. Thus, PKCι and SOX2 are genetically, biochemically, and functionally linked in LSCC, and together they drive tumorigenesis by establishing a cell-autonomous Hh signaling axis.

Ect2 links the PKCι-Par6α Complex to Rac1 Activation and Cellular Transformation

Protein kinase Cι (PKCι) promotes non-small cell lung cancer (NSCLC) by binding to Par6α and activating a Rac1-Pak-Mek1,2-Erk1,2 signaling cascade. The mechanism by which the PKCι–Par6α complex regulates Rac1 is unknown. Here we show that epithelial cell transforming sequence 2 (Ect2), a guanine nucleotide exchange factor for Rho family GTPases, is coordinately amplified and overexpressed with PKCι in NSCLC tumors. RNA interference-mediated knockdown of Ect2 inhibits Rac1 activity and blocks transformed growth, invasion and tumorigenicity of NSCLC cells. Expression of constitutively active Rac1 (RacV12) restores transformation to Ect2-deficient cells. Interestingly, the role of Ect2 in transformation is distinct from its well-established role in cytokinesis. In NSCLC cells, Ect2 is mislocalized to the cytoplasm where it binds the PKCι–Par6α complex. RNA interference-mediated knockdown of either PKCι or Par6α causes Ect2 to redistribute to the nucleus, indicating that the PKCι–Par6α complex regulates the cytoplasmic localization of Ect2. Our data indicate that Ect2 and PKCι are genetically and functionally linked in NSCLC, acting to coordinately drive tumor cell proliferation and invasion through formation of an oncogenic PKCι–Par6α-Ect2 complex.

Matrix metalloproteinase-10 is a critical effector of protein kinase Cι-Par6α-mediated lung cancer

Protein kinase Cι (PKCι) drives transformed growth of non-small cell lung cancer (NSCLC) cells through the Rho family GTPase Rac1. We show here that PKCι activates Rac1 in NSCLC cells by formation of a PKCι–Par6α complex that drives anchorage-independent growth and invasion through activation of matrix metalloproteinase-10 (MMP-10) expression. RNAi-mediated knockdown of PKCι, Par6α or Rac1 expression inhibits NSCLC transformation and MMP-10 expression in vitro. Expression of wild-type Par6α in Par6α-deficient cells restores transformation and MMP-10 expression, whereas expression of Par6α mutants that either cannot bind PKCι (Par6α-K19A) or couple to Rac1 (Par6α-ΔCRIB) do not. Knockdown of MMP-10 expression blocks anchorage-independent growth and invasion of NSCLC cells and addition of catalytically active MMP-10 to PKCι- or Par6α-deficient cells restores anchorage-independent growth and invasion. Dominant-negative PKCι inhibits tumorigenicity and MMP-10 expression in subcutaneous NSCLC tumors. MMP-10 and PKCι are coordinately overexpressed in primary NSCLC tumors, and tumor MMP-10 expression predicts poor survival in NSCLC patients. Our data define a PKCι–Par6α–Rac1 signaling axis that drives anchorage-independent growth and invasion of NSCLC cells through induction of MMP-10 expression.

The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer.

Ect2 is an oncogene in multiple human cancers. Ect2 is aberrantly overexpressed and mis-localized in multiple human tumor types, often as a result of targeted amplification of the ECT2 gene as part of the 3q26 amplicon. Ect2 is important for proliferation, migration and invasion of various types of cancer cells in vitro, and for NSCLC tumorigenicity in vivo. The role of Ect2 in cellular transformation is distinct from its physiologic role in cytokinesis, and many tumor cells appear to have evolved Ect2-independent cytokinesis mechanisms. In NSCLC cells, the ability of Ect2 to support transformation is linked to its mislocalization to the cytoplasm and activation of a Rac1-Pak-Mek1,2-Erk1,2 signaling axis that is regulated through its binding to the oncogenic PKCι/Par6α complex (Figure 4). Therefore, Ect2 and PKCι are genetically linked due to their frequent co-amplification as part of the 3q26 amplicon, and functionally and biochemically linked through formation of an oncogenic PKCι-Par6-Ect2 complex that drives transformation. Further experiments will be required to determine if Ect2 and PKCι are similarly linked in other tumors, particularly those harboring 3q26 amplification. In addition, further work is needed to elucidate the molecular mechanisms that regulate the formation, dynamics and activity of the oncogenic PKCι-Par6α-Ect2 complex. These studies hold the promise of identifying novel therapeutic approaches to cancer treatment based on inhibiting Ect2 function in cancer cells. Figure 4 Schematic diagram of the PKCι-Par6α-Ect2 oncogenic signaling axis Acknowledgments The authors wish to thank their colleagues in the Fields laboratory for helpful suggestions and critical review of the manuscript. The authors also wish to apologize to colleagues who have made important contributions to this area, but whose work could not be cited due to space limitations. The work from the Fields laboratory discussed in this article was supported by grants from the National Institutes of Health (CA081436-12), the American Lung Association/LUNGevity, the V Foundation, and the Mayo Foundation to A.P.F.

Molecular Pathways: Novel Approaches for Improved Therapeutic Targeting of Hedgehog Signaling in Cancer Stem Cells

The Hedgehog (Hh) signaling pathway is critical for embryonic development. In adult tissues, Hh signaling is relatively quiescent with the exception of roles in tissue maintenance and repair. Aberrant activation of Hh signaling is implicated in multiple aspects of transformation, including the maintenance of the cancer stem cell (CSC) phenotype. Preclinical studies indicate that CSCs from many tumor types are sensitive to Hh pathway inhibition and that Hh-targeted therapeutics block many aspects of transformation attributed to CSCs, including drug resistance, relapse, and metastasis. However, to date, Hh inhibitors, specifically those targeting Smoothened [such as vismodegib, BMS-833923, saridegib (IPI-926), sonidegib/erismodegib (LDE225), PF-04449913, LY2940680, LEQ 506, and TAK-441], have demonstrated good efficacy as monotherapy in patients with basal cell carcinoma and medulloblastoma, but have shown limited activity in other tumor types. This lack of success is likely due to many factors, including a lack of patient stratification in early trials, cross-talk between Hh and other oncogenic signaling pathways that can modulate therapeutic response, and a limited knowledge of Hh pathway activation mechanisms in CSCs from most tumor types. Here, we discuss Hh signaling mechanisms in the context of human cancer, particularly in the maintenance of the CSC phenotype, and consider new therapeutic strategies that hold the potential to expand considerably the scope and therapeutic efficacy of Hh-directed anticancer therapy. Clin Cancer Res; 21(3); 505–13. ©2015 AACR.

Matrix Metalloproteinase-10 Is Required for Lung Cancer Stem Cell Maintenance, Tumor Initiation and Metastatic Potential

Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10 −/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.

Atypical protein kinase Cι as a human oncogene and therapeutic target.

Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family. The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity. Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, nonredundant cellular functions. Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions. PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis. Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCiota has been shown to function as a bona fide oncogene, and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCiota in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCiota signaling in cancer.

Oncogenic Activity of Ect2 Is Regulated through Protein Kinase Cι-mediated Phosphorylation

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.

Protein Kinase Cα Promotes Cell Migration through a PDZ-Dependent Interaction with its Novel Substrate Discs Large Homolog 1 (DLG1)

Background: PKCα contains a unique PDZ ligand motif and is known to promote cellular migration. Results: PKCα binds and phosphorylates the scaffold DLG1; both proteins are necessary for cellular migration in non-small cell lung cancer cells. Conclusion: DLG1 coordinates PKCα signaling to promote cellular migration. Significance: Control of PKCα signaling mediated by scaffolds is crucial to promoting its downstream functions. Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.

Matrix Metalloproteinase-10 Promotes Kras-Mediated Bronchio-Alveolar Stem Cell Expansion and Lung Cancer Formation

Matrix metalloproteinase 10 (MMP-10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (Mmp10−/−) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.