Michael W. Straza

Protein Kinase Cζ Mediates Epidermal Growth Factor–Induced Growth of Head and Neck Tumor Cells by Regulating Mitogen-Activated Protein Kinase

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Therapeutic targeting of C-terminal binding protein in human cancer

The CtBP transcriptional corepressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is antagonized by p14/p19ARF tumor suppressors. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells. MTOB induced apoptosis was p53-independent, correlated with the derepression of the pro-apoptotic CtBP repression target Bik, and was rescued by CtBP over-expression or Bik silencing. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP may represent a useful therapeutic strategy in human malignancies.

Abstract B07: Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses

Progress in elucidating the molecular basis of breast cancer has allowed for treatment breakthroughs such as anti-estrogen and Her2-targeted therapy. It has also shaped the approaches to both surgical and systemic therapy. However, no similar use of molecular information has been utilized to better direct the use of radiation therapy. The development of predictive tools for the radiosensitivity of tumors could allow for personally tailored radiation doses, with treatment de-escalation for radiosensitive tumors, or dose escalation or the use of adjunct treatments in the case of radioresistant tumors. Communication between malignant tumor cells and the tumor microenvironment (TME) underlies most aspects of tumor biology, including chemotherapy and radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cell-sorting. Here we utilize these unique techniques to identify genetic variants in the TME that can affect radiation sensitivity. In CXM, human triple negative breast cancer MDA-MD-231 cells are orthotopically implanted into immunodeficient (IL2Rγ-/-) consomic rat strains, which are rat strains in which an entire chromosome is introgressed into the isogenic background of another inbred strain by selective breeding. Because the strain backgrounds are different but the tumor cells are not varied, the observed changes in tumor progression are due to genetic differences in the non-malignant TME. We hypothesized that the tumors in SS.BN3 rats (identical to SS rats but with BN strain chromosome 3) would be more sensitive to radiation due to increased tumor vascularity via CD31 staining, and increased tumor blood volume capacity, as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Our studies demonstrate differential responses to radiation in the CXM model comparing parental SS (IL2Rγ) rats to SS.BN3 (IL2Rγ) rats treated with fractionated radiation therapy (4 Gray x 3), with altered tumor growth kinetics and tumor recurrence rates. A difference was seen in time to 5-fold increase in tumor growth, with 44 vs. >130 days for SS versus SS.BN3 rats (supra-additive, p 130 days) in the SS versus SS.BN3 rats (p=0.02). These results suggest that genetic determinants in the TME affect the radiation sensitivity of genetically identical tumor cells. Using SSRS, we identified a number of candidates on rat chromosome 3 that may potentially influence radiation sensitivity by altering the tumor vasculature. Future studies will further dissect the pathways responsible for the changes in radiation sensitivity. Determining TME factors that affect the radiation sensitivity of tumors has the potential to allow for more tailored and effective radiation treatments in breast cancer. Citation Format: Carmen Bergom, Michael Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Shirng-Wern Tsaih, Howard Jacob, Michael J. Flister. Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B07.

Abstract 3678: The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma

Glioblastoma (GB) is the most aggressive malignancy affecting the central nervous system (CNS) with a median survival of 12 to 15 months even with surgery, radiation and chemotherapy. Previous research demonstrates that increased activation of NF-κB is critical for GB growth, proliferation and the up regulation of genes involved in cytokine production, cell cycle regulation, apoptosis and cell adhesion. Understanding the molecular targets that regulate NF-κB may provide more effective therapeutic targets for GB. DiRas family small GTPases, which are homologous to pro-oncogenic Ras GTPases, are tumor suppressive rather than tumor promoting and include DiRas1, DiRas2 and DiRas3 (ARHI). DiRas1 and DiRas2 have been suggested to be tumor suppressive in CNS malignancies, but the role of DiRas3 in CNS malignancies remains unknown. Here we demonstrate that expression of DiRas3 protein in GB cell lines is absent, although DiRas3 is expressed in non-malignant glial cells. Re-expression of DiRas3 in U-87 cells reduces cell proliferation by 20%. Using a NF-κB transcriptional activity luciferase reporter assay demonstrates that DiRas3 expression reduces NF-κB transcriptional activity by 70% compared to vector control. Further experiments demonstrate that decreased NF- κB activity occurs via reduced phosphorylation of the NF-κB inhibitor IκBα. The reduced phosphorylation of IκBα could be a result of decreased AKT and ERK activity, as increased ERK and AKT activity can stimulate NF-κB pathways. Our lab has previously demonstrated that the most common binding partner for DiRas1 and DiRas2 was the small GTPase binding protein SmgGDS, and DiRas1 and DiRas2 also reduce NF- κB activation. However, DiRas3 does not interact with SmgGDS, suggesting that DiRas3 can reduce NF-κB in a SmgGDS-independent manner. Understanding the role of DiRas3 and its binding partners in mediating NF- κB activation may lead to novel therapeutics for glioblastoma. Citation Format: Amy Rymaszewski, Michael Straza, Anne Frei, Carmen Bergom. The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma. [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 3678.

Abstract 1633: C-terminal binding proteins are novel drug targets

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The CtBP transcriptional corepressors have been identified as proto-oncogenes. CtBP represses pro-apoptotic genes and promotes cancer cell migration and invasion in a redox sensitive manner through a regulatory dehydrogenase domain. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and induces apoptosis in the human colorectal cancer cell line HCT116. MTOB induced apoptosis is dependent upon expression of the BH3 only protein BIK, a previously established CtBP target. Treatment of both native and transformed Mouse Embryonic Fibroblasts revealed a wide therapeutic index for CtBP inhibition. In human colon cancer cell peritoneal xenografts, MTOB treatment reduced tumor growth and induced apoptosis in vivo without notable toxicity. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Thus, CtBP is a viable therapeutic target in human cancer. In order to identify novel CtBP inhibitors, a simple dehydrogenase assay was developed that utilizes the spectrophotometric measurement of enzymatic conversion of NADH to NAD+ by the CtBP dehydrogenase activity. This assay successfully detects MTOB substrate and inhibitor activity, as well as inhibition of CtBP activity by the related compound 4-methylthio-2-hydroxybutyric acid (MTHB). This assay will be adapted for high throughput screening of small molecule libraries for novel CtBP inhibitors that can be further characterized in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2011-1633