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Supplementary Figure 2 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

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Supplementary Table 2 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

Supplementary Figure 1 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

Supplementary Table 1 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

The Ras oncoprotein may participate in driving half of human tumors and as such remains the single most significant therapeutic target in cancer. Attempts to develop targeted inhibitors of RAS have proved extremely challenging. We have developed a novel Ras binding molecule that blocks the ability of Ras to interact with its downstream effector components and thus down-regulates Ras signaling. We have also developed the first direct inhibitor of the RAS effector RALGDS. Both compounds suppresses the transforming activity of mutant forms of Ras as well as the wild type protein when it is over-active due to defects in the NF1 protein. The compounds have no detectable toxicity in animals and readily crosses the blood-brain barrier. They are potently active in xenograft models of Ras mediated transformation and can eradicate the ability of Ras addicted tumor cells to form tumors in animals. We propose the compounds may serve as the basis for the development of new treatments for oncogenic RAS driven tumors and for many Rasopathy symptoms. Citation Format: Geoffrey Justin Clark. Novel RAS inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6374.

NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21CIP1 as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21CIP1 expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21CIP1 expression and promotes proliferation. The best characterized activator of p21CIP1 is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21CIP1 via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53. [Cancer Res 2009;69(11):4629–37]

Recent advances in the study of RASSF1A, the candidate tumor suppressor gene, indicate a possible role of RASSF1A in cell cycle regulation; however, very little is known regarding molecular mechanisms underlying this control. Using small interfering RNA to knockdown endogenous RASSF1A in the breast tumor cell line HB2 and in the cervical cancer cell line HeLa, we identify that a key player in cell cycle progression, cyclin A2, is concomitantly increased at both protein and mRNA levels. In A549 clones stably expressing RASSF1A, cyclin A2 levels were diminished compared with vector control. A known transcriptional regulator of cyclin A2, p120E4F (a repressor of cyclin A2), has been shown previously by our group to interact with RASSF1A. We show that levels of p120E4F are not affected by RASSF1A small interfering RNA in HB2 and HeLa cells. However, electrophoretic mobility shift assays indicate that knockdown of endogenous RASSF1A in HB2 and HeLa cells leads to a reduction in the binding capacity of p120E4F to the cyclin A2 promoter, whereas in the A549 clone stably expressing RASSF1A the binding capacity is increased. These data are further corroborated in vitro by the luciferase assay and in vivo by chromatin immunoprecipitation experiments. Together, these data identify the cyclin A2 gene as a cellular target for RASSF1A through p120E4F and for the first time suggest a transcriptional mechanism for RASSF1A-dependent cell cycle regulation.

Activated Ras proteins interact with a broad range of effector proteins to induce a diverse series of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras may also induce growth antagonistic effects such as senescence or apoptosis. It is now apparent that some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. To date, four members of this family have been identified (Nore1, RASSF1, RASSF2, and RASSF3). We now identify a fifth member of this group, RASSF4 (AD037). RASSF4 shows approximately 25% identity with RASSF1A and 60% identity with RASSF2. RASSF4 binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells. Thus, RASSF4 appears to be a new member of the RASSF family of potential Ras effector/tumor suppressors.

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.

Dysregulation of miRNAs and chronic inflammation are strongly implicated in the development of various malignancies, including prostate cancer (PCa). Previously, our lab identified several inflammatory and immune response sequence variants in CCL5 and CCR5 significantly modified PCa risk. Recently, we propose an oncogenic role for miR-186-5p based on its up-regulation in serum from PCa patients and metastatic PCa cell lines. Moreover, miR-186-5p inhibition reduced proliferation, anchorage independent growth and invasion in metastatic PC cells (PC3, MDA-PCA-2b). We hypothesize dysregulation of inflammatory and immune response markers may enhance immune surveillance leading to a reduced aggressive tumor phenotype. Following modulations in miRNA-186 levels in normal prostate epithelial (RWPE1) and metastatic PCa (PC3) cell lines, we evaluated alterations in mRNA expression using micro-array analysis. Ectopic expression of miRNA-186 in the RWPE1 resulted in a 1.3-2.6-fold down-regulation in TLR2, IRAK2, CCL20, IL1RAP, IL1RAP, IL15, and IL17RD (FDR p-value ≥7.8x10-06). Whereas, inhibition of miR-186 in PC3 cells corresponded with a 1.4-fold up-regulation of IL18R1 (p=0.0095). Notably, these inflammatory markers are involved in cell invasion (TLR2), apoptosis (IL17RD), tumorigenesis (TLR2, IRAK2) and autoimmune diseases (CCL20). Validation of these targets is underway using qRT-PCR, western blots and luciferase reporter assays. We will assess the mechanism by which these miR-186 targets suppress aggressive cancer behavior using knock-out and knock-in cell and ultimately animal models. Future studies may identify inflammatory targets that may guide immune therapies for the effective treatment of aggressive prostate cancer. Citation Format: Suman Suman, Dominique Z. Jones-Reed, M. L. Schmidt, Geoffrey J. Clark, Carolyn Klinge, Shirish Barve, Kevin S. Kimbro, La Creis R. Kidd. Alteration of miR-186 expression modifies inflammatory markers in normal epithelial and prostate cancer cell models [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 1483. doi:10.1158/1538-7445.AM2017-1483

MicroRNA (miR) dysregulation alters the expression of cancer related genes and contributes to disease states in many cancers. For example, ectopic miR-186 expression leads to enhanced cell proliferation and migration in pancreatic cancer. However, the role of miR-186 in prostate cancer (PCa) remains unclear. Previously, we observed significant upregulation of miR-186-5p in PCa patient serum (stage III/IV) compared to controls. Furthermore, miR-186 was significantly up-regulated in metastatic PCa (PC-3) compared to normal prostate epithelial cells (RWPE1). We hypothesized miR-186 inhibition will reduce aggressive PCa in vitro. Consequently, miR-186 was transiently and stably inhibited in PC-3 cells. Cell proliferation and colony formation were evaluated for 7 and 21 days via Trypan Blue exclusion and soft agar assays. Aberrant gene expression was evaluated in transfected cells to identify miR-186 targets. Candidate miR-186 targets were selected using published reports on ‘miR-186 and cancer’, availability of robust antibodies, and statistical filtering (false discovery ≥0.05 and ±1.2 fold change). Mir-186 inhibition in PC-3 cells significantly repressed proliferation and colony formation by 34-64%. Following miR-186 inhibition in PC-3 cells, 2,343 identified mRNA targets were differentially expressed compared to scramble controls (p < 0.05). The target list was reduced to 11 up-regulated candidates. Predicted miR-186 targets are undergoing validation via qRT-PCR, western blots, and luciferase reporter assays. In addition, other studies are in progress to evaluate the impact of miR-186 on cellular proliferation, migration and invasion. Such efforts may lead to the identification of novel biomarkers to improve detection and clinical management strategies for aggressive PCa. Citation Format: Dominique Z. Jones, M. Lee Schmidt, Katharine R. Hobbing, Geoffrey Clark, LaCreis R. Kidd. Inhibition of miR-186 and repression of aggressive prostate cancer phenotype using a metastatic cell model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1906.

MicroRNAs (miRs), key regulators of cancer-related genes, are dysregulated in many solid tumors, including prostate cancer. However, the role of miR-186 in prostate cancer (PCA) requires further elucidation. Previously, we observed mature miR-186-5p was significantly up-regulated in the serum collected from PCA patients (n = 15) diagnosed with aggressive PCA (stage III/IV) disease compared to disease-free men (n = 5). In addition, miR-186 expression was significantly up-regulated in metastatic PCA cells (PC3) compared to a normal prostate epithelial cell line (RWPE1). Putative and predicted miR-186 gene targets in various cancers include pro-apoptotic and tumor suppression-related genes (e.g., P2×7, FOXO1, AKAP12). We hypothesized that miR-186 inhibition will reduce the aggressive PCA phenotype. To test this hypothesis, PC3 cells were transiently transfected with miR-186 inhibitor and scramble control for 24hrs. Cell viability and colony formation of transfected cells were evaluated via the ATPlite Luminescence assay and soft agar assay. We also evaluated expression profiles of predicted miR-186 gene targets in PCA cell lines and transiently transfected PC3 cells using qRT-PCR. Transient inhibition of miR-186 repressed cellular proliferation and colony formation by 48-58% in 72-96hrs and 30% after 21 days, respectively. We observed a 1.6-2.24 fold up-regulation in ROCK1 gene expression in PC3 cells treated with miR-186 inhibitor. However, additional studies are needed to assess whether stable inhibition of miR-186 will increase PCA invasiveness by up-regulating ROCK1 using in vitro assays and murine models. Moreover, predicted miR-186 gene targets require validation using qRT-PCR, western blots and luciferase reporter assays. Such efforts may lead to the identification of novel biomarkers to improve diagnostic, prognostic and clinical management strategies. Citation Format: Dominique Zilpha Jones, Katharine R. Hobbing, M. L. Schmidt, Geoffrey J. Clark, LaCreis R. Kidd. MicroRNA-186 inhibition alters cell proliferation and colony formation in prostate cancer. [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 3085. doi:10.1158/1538-7445.AM2015-3085

In addition to driving growth and transformation, activated forms of Ras are potent inducers of oncogene induced senescence (OIS). OIS appears to be a major barrier that must be overcome to permit Ras driven tumorigenesis. The signaling pathways utilized by Ras to induce senescence, and how they are subverted during tumor development remain poorly characterized. NORE1A is a member of the RASSF family of tumor suppressors. It binds directly to activated Ras via the Ras effector domain and acts as a Ras death effector. Frequent loss of NORE1A expression is observed in many tumor types and hereditary genetic defects in NORE1A predispose carriers to cancer. We have found that NORE1A can connect Ras to the induction of p21CIP1 and cell cycle arrest. Therefore, we sought to determine if NORE1A might play a role in Ras induced senescence. We now show that NORE1A is a potent mediator of Ras induced senescence. Knockdown of NORE1A suppresses the ability of Ras to induce senescence in multiple cell systems and enhances Ras driven transformation. We have identified two novel signaling pathways activated by Ras/NORE1A. First, NORE1A forms a Ras regulated, endogenous complex with the kinase HIPK2. HIPK2 can phosphorylate p53 to induce apoptosis or recruit acetyltransferases to acetylate p53 to induce senescence signaling. NORE1A suppresses HIPK2 apoptotic post-translational modifications of p53 but activates pro-senescence post-translational modifications. NORE1A also binds and destabilizes mdm2 to enhance the stability of nuclear p53. Primary human tumors show a close correlation between the expression levels of NORE1A and acetylated p53. In addition to p53, we show that NORE1A also links Ras to the regulation of Retinoblastoma (Rb) protein. NORE1A promotes a potent, Ras dependent stabilization of the Rb protein. It also promotes dephosphorylation of RB, an activating event. Thus, NORE1A is a double-barreled Ras senescence effector which connects Ras to two of the most important senescence regulating tumor suppressors in the cell. Loss of NORE1A activity in tumors is usually due to epigenetic inactivation or aberrant protein degradation by calpains. Both of these mechanisms, in principal, may be subject to clinical intervention to restore NORE1A function. This may provide a novel approach to antagonizing Ras driven tumors. Citation Format: Howard Donninger, Diego Calvisi, Thibaut Barnoud, M. Lee Schmidt, Geoffrey J. Clark. NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A06. doi: 10.1158/1557-3125.RASONC14-A06