Tiantian Cui

Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells.

Significance Cancer stem cells (CSCs) exhibit enhanced chemo/radiotherapy resistance, and their survival following cancer treatment is believed to be responsible for tumor recurrence and metastasis. Thus, understanding the mechanisms through which CSCs survive conventional chemotherapy is essential for identification of new therapeutic strategies to prevent tumor relapse. Our findings that ovarian CSCs survive cisplatin treatment through elevated expression of polymerase η represent an opportunity to eradicate CSCs and improve the survival of ovarian cancer patients. In addition, identification of miR-93 as the regulator of polymerase η expression provides a target to increase the efficacy of cisplatin treatment. Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.

miR-93 promotes TGF-β-induced epithelial-to-mesenchymal transition through downregulation of NEDD4L in lung cancer cells.

The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-β-mediated EMT in lung cancer cells. miR-93 binds directly to the 3′-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-β signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-β treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-β-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.

Differential DNA lesion formation and repair in heterochromatin and euchromatin

Discretely orchestrated chromatin condensation is important for chromosome protection from DNA damage. However, it is still unclear how different chromatin states affect the formation and repair of nucleotide excision repair (NER) substrates, e.g. ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts (6-4PP), as well as cisplatin-induced intrastrand crosslinks (Pt-GG). Here, by using immunofluorescence and chromatin immunoprecipitation assays, we have demonstrated that CPD, which cause minor distortion of DNA double helix, can be detected in both euchromatic and heterochromatic regions, while 6-4PP and Pt-GG, which cause major distortion of DNA helix, can exclusively be detected in euchromatin, indicating that the condensed chromatin environment specifically interferes with the formation of these DNA lesions. Mechanistic investigation revealed that the class III histone deacetylase SIRT1 is responsible for restricting the formation of 6-4PP and Pt-GG in cells, probably by facilitating the maintenance of highly condensed heterochromatin. In addition, we also showed that the repair of CPD in heterochromatin is slower than that in euchromatin, and DNA damage binding protein 2 (DDB2) can promote the removal of CPD from heterochromatic region. In summary, our data provide evidence for differential formation and repair of DNA lesions that are substrates of NER. Both the sensitivity of DNA to damage and the kinetics of repair can be affected by the underlying level of chromatin compaction.

DDB2 modulates TGF-β signal transduction in human ovarian cancer cells by downregulating NEDD4L

The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.

The role of desmosomes in carcinogenesis

Desmosomes, which are intercellular adhesive complexes, are essential for the maintenance of epithelial homeostasis. They are located at the cell membrane, where they act as anchors for intermediate filaments. Downregulation of desmosome proteins in various cancers promotes tumor progression. However, the role of desmosomes in carcinogenesis is still being elucidated. Recent studies revealed that desmosome family members play a crucial role in tumor suppression or tumor promotion. This review focuses on studies that provide insights into the role of desmosomes in carcinogenesis and address their molecular functions.

DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1

Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that the CSC subpopulation in ovarian cancer can be limited by DNA damage-binding protein 2 (DDB2). Here, we demonstrated that the ovarian CSC subpopulation can be maintained via cancer cell dedifferentiation, and DDB2 is able to suppress this non-CSC-to-CSC conversion by repression of ALDH1A1 transcription. Mechanistically, DDB2 binds to the ALDH1A1 gene promoter, facilitating the enrichment of histone H3K27me3, and competing with the transcription factor C/EBPβ for binding to this region, eventually inhibiting the promoter activity of the ALDH1A1 gene. The de-repression of ALDH1A1 expression contributes to DDB2 silencing-augmented non-CSC-to-CSC conversion and expansion of the CSC subpopulation. We further showed that treatment with a selective ALDH1A1 inhibitor blocked DDB2 silencing-induced expansion of CSCs, and halted orthotopic xenograft tumor growth. Together, our data demonstrate that DDB2, functioning as a transcription repressor, can abrogate ovarian CSC properties by downregulating ALDH1A1 expression.

DDB2 increases radioresistance of NSCLC cells by enhancing DNA damage responses

Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2 in NSCLC could be used as a biomarker to predict radiosensitivity of the patients. Targeting Chk1 can be used to increase the efficacy of radiotherapy in patients of NSCLC possessing high levels of DDB2.

Depleting ovarian cancer stem cells with calcitriol

Cancer stem cells (CSCs) represent the root of many solid tumors including ovarian cancer. Eradication of CSCs represents a novel cancer therapeutic strategy. Calcitriol, also known as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an active metabolite of vitamin D, functioning as a potent steroid hormone. Calcitriol has shown anti-tumor effects in various cancers by regulating multiple signaling pathways. It has been reported that calcitriol can regulate the properties of normal and CSCs. However, the effect of calcitriol on the ovarian cancer growth and ovarian CSCs is still unclear. Here, by using a mouse subcutaneous xenograft model generated with human ovarian cancer cells, we have demonstrated that administration of calcitriol is able to strikingly delay the tumor growth. Calcitriol treatment can also deplete the ovarian CSC population characterized by ALDH+ and CD44+CD117+; decrease their capacity to form sphere under the CSC culture condition, and reduce the frequency of tumor-initiating cells, as evaluated by in vivo limiting dilution analysis. Mechanistic investigation revealed that calcitriol depletes CSCs via the nuclear vitamin D receptor (VDR)-mediated inhibition of the Wnt pathway. Furthermore, the activation of VDR pathway is more sensitive to calcitriol in ovarian CSCs than in non-CSCs, although the expression levels of VDR are comparable. Taken together, our data indicate that calcitriol is able to deplete the ovarian CSC population by inhibiting their Wnt signaling pathway, consequently, impeding the growth of xenograft tumors.

Abstract 4784: Regulation of ovarian cancer stem cell population by DDB2

Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that DNA damage-binding protein 2 (DDB2) is able to reduce the abundance of CSCs in the bulk ovarian cancer cells, providing a novel mechanism to explain the DDB2-mediated suppression of tumorigenicity and metastasis, and also suggesting that low expression of DDB2 is required for the maintenance of CSCs. However, the underlying mechanisms still remain unclear. By using the Tet-On DDB2 modulation system, we have confirmed our previous finding that downregulation of DDB2 expands the CSC population in the 2008 ovarian cancer cell line. We also found that DDB2 is able to suppress non-CSC-to-CSC conversions in this cancer cell line. DDB2 has been recognized as a transcriptional regulator. Our microarray analysis has identified ALDH1A1 to be targeted and regulated by DDB2. The downregulation of ALDH1A1 expression by DDB2 at both mRNA and protein levels has been validated in various ovarian cancer cell lines. The mechanistic investigation demonstrated that DDB2 can bind to the promoter region of the ALDH1A1 gene, facilitating the enrichment of histone H3K27me3 by recruiting EZH2 to the promoter region, eventually inhibiting the promoter activity of the ALDH1A1 gene. In addition, we also found that DDB2 competes with transcription factor C/EBPβ for binding to the ALDH1A1 promoter, indirectly inhibiting the ALDH1A1 promoter activity. Finally, we knocked down the expression of DDB2 and ALDH1A1 individually or simultaneously in the 2008 ovarian cancer cell line, and analyzed their tumorigenicity. We found that downregulation of ALDH1A1 is able to block DDB2 silencing-induced expansion of the CSC population, indicating that ALDH1A1 plays a critical role in DDB2-mediated suppression of the CSC population in ovarian cancer cells. In summary, our data demonstrated that DDB2, functioning as a transcription repressor, is able to abrogate ovarian CSC properties by downregulating ALDH1A1 expression. This study provides a novel mechanism underlying the regulation of the CSC population, and would facilitate the development of efficient strategies for eliminating CSCs to prevent tumor relapse and metastasis in ovarian cancers. (Supported by NIH R01CA151248) Citation Format: Tiantian Cui, Amit Kumar Srivastava, Chunhua Han, Zhiqin Gao, Xiaoli Zhang, Altaf A. Wani, Qi-En Wang. Regulation of ovarian cancer stem cell population by DDB2 [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 4784. doi:10.1158/1538-7445.AM2017-4784

Abstract 5726: A novel tumor-promoting role for miR-4516 in glioblastoma

Background: Glioblastomas are the most aggressive high-grade brain tumors, which are often life-threatening due to their location and rapid growth. Although survival rates differ depending on a variety of genetic and environmental factors, the average survival rate for a glioblastoma (GBM) patient is less than 15 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. Here we demonstrate a novel role for miR-4516 in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods: Formalin-fixed, paraffin-embedded tissue blocks (n=268) were collected for all patients and total RNA was isolated. miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle,WA). Functional characterization studies were conducted in vitro and in vivo. The effect of miR-4516 on GBM cell growth and motility were evaluated by cell proliferation assay, migration and invasion assay, and Annexin-V assay. Realtime PCR, immunoblotting, and 3’ untranslated region luciferase assays were used to analyze miR-4516 targets and signaling pathways. Intracranial injection will be performed to investigate the role of miR-4516 in tumor growth in vivo. Results: Univariate analysis showed that miR-4516 expression in GBM patients was inversely correlated with overall survival (FDR=0.002, p=1.02E-05). Knockdown of miR-4516 blocked tumor growth and induced cell apoptosis. Tumor cell growth, migration and invasion were induced in both transient miR-4516 overexpressed GBM cells (LN229, LN18, and U87) and stable miR-4516 overexpressed GBM cells (U87-EGFRvIII). These miR-4516 tumor-promoting effects were mediated in part via direct targeting PTPN14 and CDKN1A. Investigation of the other miR-4516 targets and in vivo functional study are in process. Conclusion: Taken together, these results suggest that miR-4516 acts as a prognostic biomarker for GBM patients. Funding Information: 1R01CA169368 (PI: Houghton; Co-I:Chakravarti); 1R01CA11522358 Multiple-PI R01: Chakravarti (PI); Xia (PI); 1R01CA1145128 Baroukhim (PI); Chakravarti (Co-PI) 7/2015-6/2020; R01CA108633 (PI:Chakravarti); 1RC2CA148190 (Scientific PI: Chakravarti) Citation Format: Tiantian Cui, Ashley Gray, Ziyan Liu, Marjolein Geurts, Pierre Robe, Joseph McElroy, Erica Hlavin Bell, Arnab Chakravarti. A novel tumor-promoting role for miR-4516 in glioblastoma [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 5726. doi:10.1158/1538-7445.AM2017-5726