Ji-Eun Oh

A CDK4/6 inhibitor enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring mutant KRAS as well as wild-type KRAS

The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15–30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma.

Synergistic antitumor efficacy of sequentially combined paclitaxel with sorafenib in vitro and in vivo NSCLC models harboring KRAS or BRAF mutations.

Studies on non-small cell lung cancer (NSCLC) patients with KRAS or BRAF mutations are urgently needed to improve clinical outcomes. We evaluated the cytotoxicities of paclitaxel and sorafenib alone and in combination in NSCLC cell lines with KRAS or BRAF mutations and investigated the mechanism of the interaction between the drugs. We found synergistic antitumor efficacy with paclitaxel followed by sorafenib in in vitro and in vivo models of NSCLC. And, we determined that downregulation of the phosphorylated ERK and Rb, and Mcl-1 plays a critical role in the synergistic activity of the drugs. Further clinical trials are needed to verify the antitumor efficacy of this combination.

Molecular genetic characterization of p53 mutated oropharyngeal squamous cell carcinoma cells transformed with human papillomavirus E6 and E7 oncogenes

Patients with HPV-positive oropharyngeal cancer show better tumor response to radiation or chemotherapy than patients with HPV-negative cancer. HPV oncoprotein E6 binds and degrades a typically wild-type p53 protein product. However, HPV16 infection and p53 mutation infrequently coexist in a subset of HNSCCs. The purpose of this study was to investigate the mechanisms through which tumor biology and molecular genetic mechanisms change when two HPV-negative, p53-mutated oropharyngeal cell lines (YD8, non-disruptive p53 mutation; YD10B, disruptive p53 mutation) derived from patients with a history of heavy smoking are transfected with HPV E6 and E7 oncogenes in vitro. Transfection with HPV E6 and E7 oncogenes in YD8, reduced the abundance of proteins encoded by tumor suppressor genes, such as p-p53 and p-Rb. Cell proliferative activity was increased in the cells transfected with E6E7 compared to cells transfected with vector alone (P=0.09), whereas the invasiveness of E6E7-transfected cells was significantly reduced (P=0.02). cDNA microarray of the transfected cells with E6E7 showed significant changes in mRNA expression in several signaling pathways, including focal adhesion, JAK-STAT signaling pathway, cell cycle and p53 signaling pathway. Regarding the qPCR array for the p53 signaling pathway, the mRNA expression of STAT1 was remarkably upregulated by 6.47-fold (P<0.05); in contrast, IGF-1R was significantly downregulated by 2.40-fold in the YD8-vector compared toYD8-E6E7 (P<0.01). Finally, data collected from these two array experiments enabled us to select two genes, STAT1 and IGF-1R, for further study. In immunohistochemical study, nuclear STAT1 expression was slightly higher in HPV-positive compared to HPV-negative oropharyngeal tumors (P=0.18); however, cytoplasmic STAT1 was significantly lower in HPV-positive cases (P=0.03). IGF-1R expression levels were remarkably lower in HPV-positive compared to HPV-negative cases (P=0.01). Our data suggest that upregulated STAT1 and interferon signals by HPV16 E6 and E7 genes may play a major role in the relatively favorable prognosis for HPV-positive oropharyngeal squamous cell carcinoma cases with non-disruptive p53 mutations.

Abstract 1291: Tumor growth inhibitory effect in EIF4B silenced in vitro and in vivo lung cancer models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Backgraound The eIF4B, a key molecule for initiation of protein translation, play an important role in cell growth, proliferation and survival. We investigated that eIF4B is associated with the molecular mechanism of de novo resistance against EGFR TKIs and its role on cell proliferation and survival in NSCLC cells. Method Among five NSCLC cell lines harboring EGFR and K-RAS wild type, PC14 cells showing the highest IC50, were selected for our experiment. In eIF4B silenced PC14 cells by shRNA, we evaluated cell proliferation by direct cell counts and SRB assay and cell cycle distribution using flow cytometry. The expression of signal molecules, transcription factors and apoptosis associated proteins was measured by Western blot. In vivo tumor model established by subcutaneous injection of eIF4B silenced PC14 cells, the retardation of tumor growth was measured. Results In PC14 cells transformed with eIF4B shRNA, we observed the protein expression of eIF4B was reduced (>70% at day 5). The proliferation of eIF4B silenced cells was greatly inhibited both in vitro and in vivo tumor model. Between eIF4B silenced cells and control cells, there was no significant difference in anti-proliferative activity of several target inhibitors (enzastaurin, gefitinib, LY294002, vandetanib) against EGFR-PI3K-AKT signaling molecules. The expression of proteins involved in cell survival (pEGFR, pAKT and mTOR), transcription factors (pNFkB and TS) and anti-apoptotic protein (bcl-2), was decreased and apoptosis associated proteins (phospho-p53 and active caspase-3) were increased. The G0/G1 cell fraction in eIF4B silenced cells was reduced (47.3% vs. 55.1% in control cells) and accumulation of apoptotic cells was observed (12.7% vs. 1.5% in control cells; shown as sub G1 population). Conclusion Our results demonstrated that eIF4B silencing resulted in a marked decrease of several anti-apoptotic and pro-proliferative proteins and eIF4B was required for cell proliferation and survival by regulating cell cycle and cell signaling molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1291. doi:1538-7445.AM2012-1291

Abstract 185: Alteration of microRNA expression profiles in HPV-associated squamous cell carcinoma of the head and neck (SCCHN)

Background High-risk type of human papillomavirus (HPV) has recently been found to be etiologically associated with 20 to 25% of SCCHN, particularly common in or pharyngeal tumors. We investigated the protein expression of PTEN/PI3K/AKT/mTOR signal pathway on HPV 16 +/− tonsillar squamous cell carcinomas. And, we analyzed microRNA expression profile in tongue cancer cell line transfected with HPV 16 E6/E7 oncogene. Method Tonsil cancer patients who had been treated at Seoul St. Mary9s Hospital between 2000 and 2009 were reviewed. Tissue microarrays were prepared from formalin-fixed, paraffin-embedded tumor tissues of 65 surgical specimens, and stained with antibodies against PTEN, PIK3CA, phosphorylated Akt Ser473 , and phosphorylated mTOR Ser2448 . High-risk HPV infection status was determined by in situ hybridization. The protein expression of EGFR, PIK3CA, pAkt, mTOR, and PTEN was assessed by immunohistochemistry. MicroRNA expression was studied using microarrays (PANArray™ miRNA expression profiling kit) in two tongue cancer cell lines (YD8 and YD10B) transfected with HPV 16 E6/E7 oncogene. Results Of the 65 pts, 26 (40%) pts were HPV positive. Overall survival and relapse-free survival were significantly different between two groups (P = 0.05 and P = 0.036, respectively). PTEN expression was significantly higher in HPV-positive- than in HPV-negative patients (P = 0.02). PIK3CA, phosphorylated Akt Ser473 , and phosphorylated mTOR Ser2448 expression was negatively correlated with HPV status, yet not significantly. HPV status (P = 0.03) and nodal stage (P = 0.05) were found to significantly affect overall survival, as determined by multivariate analysis. The expression profile of miRNAs was different in HPV 16 E6/E7 transformed cells compared with vector alone cells. In the final round, 8 of 158 miRNAs were selected to be most differently expressed miRNAs; let-7b, miR-1, miR-107, miR-122, miR-127-5p, miR-95, miR-23b and miR-370 (>1.2-fold). Conclusion We observed the higher expression of PTEN in HPV 16 positive tonsil tumor than HPV 16 negative tumor. Our data suggest that some microRNAs induced by E6/E7 oncogene might directly affect PTEN expression level independent of p53. We are underway on the experiments confirming that PTEN is regulated by which among these miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 185. doi:1538-7445.AM2012-185

Abstract A66: Antiproliferative effects of CDK4/6 inhibition alone and combination with chemotherapy in KRAS-mutated NSCLC.

Background: Deregulation of the p16INK4A-cyclin D-cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in various solid tumors and represents one of the attractive targets in cancer treatment. KRAS mutation confers intrinsic resistance to cytotoxic chemotherapeutic agents, and to EGFR TKIs leading to treatment failure. An effective systemic treatment for adenocarcinomas harboring KRAS mutations are urgently needed to improve clinical outcomes. Purpose: CINK4, a chemical inhibitor of cyclin-dependent kinase 4, has demonstrated effective anti-proliferative activity in vivo in the presence of pRb. Herein, we investigated that CINK4 may increase its anti-proliferative activity in the NSCLC cells harboring KRAS mutation Methods: We measured the anti-proliferative activities of CINK4 in A549 (KRAS G12S ), H358 (KRAS G12C ), PC14 (KRAS wild ), and Calu-3 (KRAS wild ) cell lines using SRB assay, cell cycle distribution quantified by flow cytometry, and the expression of cell cycle regulators and apoptosis-related proteins was evaluated by Western blotting. Multiple drug effect was analyzed to study an interaction between two drugs. The nature of the interaction between two agents was calculated for the combination index (CI). Results: Concentration- and time-dependent anti-proliferative effects of CINK4 (0.1∼40 μM concentration range) and pacitaxel (0.1∼300 nM) alone were seen in four NSCLC cell lines, and were not significantly different between each cell line (48 h IC 50 : 10.4 ± 0.2 μM and 5.5 ± 0.9, 10.1± 1.9 μM and 5.2 ± 0.9, 6.6 ± 0.8 μM and 3.6 ± 1.0, and 8.4 ± 0.2 μM and > 10 nM, in A549, H358, PC14, and Calu-3, respectively). After 48 h CINK4 treatment alone, the G0/G1phase cell population increased with increment of subG1 faction in concentration-dependent manner. CINK4 reduced cyclinD1 and Rb phosphorylation and increase the expression of cleaved PARP in concentration-dependent manner in A549, H358, and PC14 cell lines. And CINK4 combined with paclitaxel in two KRAS mutated cell lines showed enhanced anti-proliferative activity of paclitaxel. The CI values of CINK4 and palitaxel combination in A549 and H358 were 0.51 ± 0.16 and 0.63± 0.12, respectively. Conclusions: CINK4 showed promising anti-tumor activity in NSCLC cell lines. NSCLC harboring KRAS mutation may have clinical benefit from CDK4/6 inhibitor in combined with chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A66.