Erhong Meng

ALDH1A1 Maintains Ovarian Cancer Stem Cell-Like Properties by Altered Regulation of Cell Cycle Checkpoint and DNA Repair Network Signaling

Objective Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. Methods Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. Results ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. Conclusion This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

Hedgehog signaling pathway regulates the growth of ovarian cancer spheroid forming cells

The hedgehog (Hh) pathway has been shown to be activated in numerous malignancies as well as in cancer stem cells. We sought to determine the importance of the Hh pathway in regulating growth and development of ovarian cancer spheroid-forming cells (SFCs). Ovarian cancer cell lines (ES2, TOV112D, OV90, and SKOV3) as well as a normal ovarian epithelial cell line (IOSE80) were grown in non-adherent growth conditions to form SFCs. Western blot analysis was used to determine the expression of Hh pathway proteins SMOH, PTCH, GLI1. SFCs were treated with Hh agonists (SHH and IHH) as well as an Hh inhibitor (cyclopamine) to determine changes in spheroid growth and survival. All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions while IOSE80 failed to form SFCs. Compared to IOSE80, ovarian cancer cell lines demonstrated significant activation of the Hh pathway as defined by increased expression of intranuclear GLI1. Both Hh agonists demonstrated significant increases in spheroid volume of at least 42-fold for SHH-treated cells and 46-fold for IHH-treated cells. With regard to survival, SFCs were 30-50% more resistant to cyclopamine than their corresponding monolayer cells. Despite this resistance, inhibition of the Hh pathway with cyclopamine prevented further growth of SFCs with a 10-, 5-, and 4-fold restriction in growth for ES2, SKOV3, and TOV112D, respectively. The hedgehog pathway appears to be important in regulating growth of ovarian cancer spheroid-forming cells. The activation and inhibition of this pathway demonstrates significant correlation to enhanced growth and growth restriction, respectively.

Targeted inhibition of telomerase activity combined with chemotherapy demonstrates synergy in eliminating ovarian cancer spheroid-forming cells

OBJECTIVE Telomerase activity (TA) is often used as a molecular marker for cancer aggressiveness. Our objectives were to determine the TA in ovarian cancer cell lines and the effectiveness of targeting telomerase for cancer therapy. METHODS Ovarian cancer cell lines of various histologic subtypes were chosen to correspond to decreasing levels of clinical aggressiveness. Cells were grown in non-adherent growth conditions to form spheroid-forming cells (SFC). Telomerase activity was quantified using the TRAPeze RT Telomerase Detection Kit and confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Cell proliferation survival assays were performed after treatment with a small molecule telomerase inhibitor BIBR1532 both with and without multiple chemotherapeutic agents. RESULTS Compared to monolayer, TA from SFC correlated to the innate clinical aggressiveness of ovarian cancer cell lines ES2, SKOV3, and TOV112D. Treatment with BIBR1532 resulted in up to a 12-fold decrease in TA compared to controls. SFCs were significantly more resistant to BIBR1532 compared to monolayer cell lines; however, it showed reasonable efficacy at 100 uM. In combination assays, the addition of BIBR1532 to carboplatin yielded the most favorable results in regards to synergy in all three cell lines evaluated. CONCLUSIONS Telomerase activity appears to correlate to the clinical aggressiveness seen in histologic subtypes of ovarian cancer. BIBR1532 demonstrated significant inhibition of TA as well as reasonable efficacy as a single agent. Inhibition of telomerase with BIBR1532 in combination with carboplatin demonstrated a more than additive effect in-vitro and could represent a novel targeted therapy for ovarian cancer.

The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

Loss of tumor suppressor Merlin results in aberrant activation of Wnt/β-catenin signaling in cancer

The expression of the tumor suppressor Merlin is compromised in nervous system malignancies due to genomic aberrations. We demonstrated for the first time, that in breast cancer, Merlin protein expression is lost due to proteasome-mediated elimination. Immunohistochemical analysis of tumor tissues from patients with metastatic breast cancer revealed characteristically reduced Merlin expression. Importantly, we identified a functional role for Merlin in impeding breast tumor xenograft growth and reducing invasive characteristics. We sought to determine a possible mechanism by which Merlin accomplishes this reduction in malignant activity. We observed that breast and pancreatic cancer cells with loss of Merlin show an aberrant increase in the activity of β-catenin concomitant with nuclear localization of β-catenin. We discovered that Merlin physically interacts with β-catenin, alters the sub-cellular localization of β-catenin, and significantly reduces the protein levels of β-catenin by targeting it for degradation through the upregulation of Axin1. Consequently, restoration of Merlin inhibited β-catenin-mediated transcriptional activity in breast and pancreatic cancer cells. We also present evidence that loss of Merlin sensitizes tumor cells to inhibition by compounds that target β-catenin-mediated activity. Thus, this study provides compelling evidence that Merlin reduces the malignant activity of pancreatic and breast cancer, in part by suppressing the Wnt/β-catenin pathway. Given the potent role of Wnt/β-catenin signaling in breast and pancreatic cancer and the flurry of activity to test β-catenin inhibitors in the clinic, our findings are opportune and provide evidence for Merlin in restraining aberrant activation of Wnt/β-catenin signaling.

GLI1 upregulates C-JUN through a specific 130-kDa isoform

The Hedgehog pathway is molecularly linked to increased resistance to cisplatin and increased repair of platinum-DNA damage, through C-JUN. GLI1, which has five known isoforms, is a positive transcriptional regulator in Hedgehog. Southwestern blot assay, EMSA and ChIP assays indicate that only one of five isoforms of GLI1 may be responsible for the Hedgehog link with C-JUN and thus, increased platinum-DNA adduct repair. Cancer tissues express this 130-kDa isoform at levels 6-fold higher than non-malignant tissues; and this isoform exists in abundance in six of seven ovarian cancer cell lines examined.

Abstract 957: Aldehyde dehydrogenase isoform 1A1 mediates platinum resistance in ovarian cancer through the KLF4/p21 cell cycle arrest pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Aldehyde dehydrogenase isoform 1A1 (ALDH1A1) activity has been reported in numerous cancers to have a role in chemoresistance. However, the previously proposed resistant mechanism of protecting DNA via free radical scavenger and thus promoting cell survival within a chemically hostile environment has yet to be elicited in cancer. Considering the heterogeneity of cancer, we propose an alternative mechanism of ALDH-mediated platinum-resistance in ovarian cancer must exist. Methods: Isogenic ovarian cancer cell lines (A2780: platinum sensitive & A2780/CP70: platinum resistant) were evaluated for potential mechanisms of platinum resistance. ALDEFLUOR kit was utilized to measure ALDH activity as well as sort cell lines into ALDH(+) & ALDH(-) phenotypes for experiments. An ALDH1A1 shRNA knockdown model was established to determine its effect on cancer stem cell properties, downstream signaling, and platinum resistance. RTQ, Western blot and PCR microarrays were utilized to evaluate potential mechanisms of ALDH-mediated platinum resistance, with select downstream siRNA mediated downregulation of KLF4. Results: Compared to Platinum-sensitive A2780 cells, platinum-resistant CP70 cells demonstrated a 100-fold increase in ALDH1A1 activity (0.2 vs. 24%, respectively (p< 0.001). ALDH(+) cells demonstrated enhanced malignant characteristics of 2-fold increase of tumorigenicity and invasion. A 3-fold increase in cancer stem cell transcriptional factor Kruppel-like factor-4 (KFL4) was shown in ALDH(+) cells (p=0.007). Increased KFL4, upregulated cell-cycle regulator p21, thereby causing G1 arrest and allows for cancer cell repair and survival. Compared to control, shRNA-ALDH knockdown demonstrated expected decreases in KFL4, with subsequent 5-fold decrease in p21 levels and its associated mediator CDK4. Cell cycle kinetics demonstrated a significant decrease in G1-associated cell cycle arrest with decreases in apoptotic threshold, which allows DNA damage to induce apoptosis confirmed with 4-fold increase in apoptotic factor BAX and cell death. Platinum sensitivity was restored in shRNA-ALDH cells, with significant increases in response rates to carboplatin with a 60% reduction of carboplatin dose needed to achieve IC50. Conclusion: ALDH mediates platinum resistance via a specific KFL4/p21 cell cycle arrest pathway. Inhibition of this pathway resulted in reversal of platinum resistant ovarian cancer cells and could be utilized for possible targeting for inducing platinum sensitivity. Citation Format: Erhong Meng, Aparna Mitra, Steven McClelan, Lalita Shevde, Rodney P. Rocconi. Aldehyde dehydrogenase isoform 1A1 mediates platinum resistance in ovarian cancer through the KLF4/p21 cell cycle arrest pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 957. doi:10.1158/1538-7445.AM2013-957 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5613: A specific isoform of Gli1 binds the Gli-binding site of the c-jun and c-fos promoters

Gli1 participates in the transcriptional regulation of c-jun, which in turn, participates in the transcriptional regulation of genes in nucleotide excision repair. The c-jun promoter and the c-fos promoter have identical Gli-binding-sites that bind Gli1. C-jun and C-fos form the transcriptionally active heterodimer Activator Protein 1, AP1. AP1, is the positive transcriptional regulator for ERCC1, and other DNA repair proteins. Gli1 is a transcription factor in the Hedgehog pathway, and there are five known isoforms of the Gli1 protein that exist in human cells. We have investigated whether there is a specific isoform of Gli1 that binds to the transcriptional regulatory sequences of c-jun and of c-fos. Detailed studies were performed in cisplatin-resistant A2780-CP70 human ovarian cancer cells. EMSA studies demonstrated the presence of a Gli protein in these cells which bind to the Gli-binding-site, as well as the consensus Gli-binding sequence. Supershift EMSA assays show that Gli1 binds the Gli binding sites of c-jun and of c-fos. Southwestern blot analyses of protein lysates from A2780-CP70 cells demonstrated that only one of the five known Gli1 protein isoforms, the 130 kDa, bind the Gli-specific binding site in the promoter of c-jun and c-fos. No Gli2 protein binds this specific binding site in the c-jun promoter, in these cells. To further confirm the 130 kDa Gli1 isoform was responsible for binding the c-jun promoter, the full-length Gli1 with a C-terminal myc tag was transfected into cells, and the protein products were assessed by immunoprecipitation and Southwestern blot analysis. The transfected full length Gli1-myc generated a 130 kDa protein that binds the Gli1-specific binding site in the promoter of c-jun. The presence of this 130 kDa Gli1 isoform was also documented in: six additional human ovarian cancer cell lines, and ten clinical ovarian tissue samples. Seven were ovarian cancer tissue and three were non-cancer ovarian tissues. The 130 kDa Gli1 isoform was present in all specimens examined, but protein levels were six-fold higher in malignant tissues. We conclude that the 130 kDa isoform of Gli1 bind the Gli-binding site in the promoters of c-jun and c-fos. Therefore, Gli1 may be the transcriptional regulator of c-jun and c-fos, and thereby regulates the transcription of ERCC1 and genes of nucleotide excision repair. 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 5613. doi:1538-7445.AM2012-5613

Abstract 3334: Telomerase activity corresponds to the level of aggressiveness of ovarian cancer stem cells and are a potential site of targeted therapy

Objective: Cancer stem cells possess the important property of self-renewal. Telomerase activity (TA) is often used as a molecular marker for cancer aggressiveness and allows propagation of cancer cells by adding telomere repeats which can result in unlimited cell replication. As such, the potential of telomerase in maintaining self-renewal properties of cancer stem cells have been proposed. Our objectives were to determine the TA of ovarian cancer stem cells (OSC) and the effectiveness of targeting telomerase for cancer therapy. Methods Ovarian cancer cell lines (ES2, TOV112D, and SKOV3) were grown in non-adherent growth conditions to form spheroid forming cells for numerous successive spheroid generations. Flow cytometry determined the CD44+/CD24- phenotype consistent with cancer stem-cells. Histologic subtypes were chosen to correspond to decreasing levels of aggressiveness: ES2- clear cell; SKOV3- papillary serous; and TOV112D- endometrioid. TRAPEZE RT Telomerase Detection Kit utilized flourescently labeled primers to detect and quantify telomerase activity by RT-PCR. Results were confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Each cancer cell line was treated with 10, 20, 50, and 100uM of a small molecule telomerase inhibitor BIBR1532 measured by cell proliferation survival assay for a total of 72 hours. Results All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions with flow cytometry demonstrating the enrichment of cancer stem cell phenotype: CD44+/CD24-. TA was significantly increased in spheroid forming cells enriched for OSC in both ES2 (5-fold increase) and SKOV3 (6-fold increase) (p Conclusions Telomerase activity appears to increase with ovarian cancer stem cells and supports the hypothesis that it could be important in the self-renewal properties of cancer stem cells. Inhibition of telomerase with a competitive small molecule demonstrated significant response in-vitro and could represent novel targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3334.