Fanliang Meng

Anticancer efficacy of cisplatin and trichostatin A or 5-aza-2′-deoxycytidine on ovarian cancer

Background:To evaluate the anticancer efficacy of the combination of epigenetic modifiers and cisplatin in human ovarian cancer.Methods:The effect of trichostatin A (TSA) and 5-aza-2′-deoxycytidine alone or in combination with low-dose cisplatin was evaluated on human ovarian cancer cell lines in vitro. We measured drug interaction by MTS assay, migration by transwell assay, expression of epithelial to mesenchymal transition (EMT) markers (Twist, Snail, Slug, E-cadherin, and N-cadherin), pluripotency markers (Oct4, Sox2, and Nanog), and epigenetic markers (DNMT3A, LSD1 and H3K4me2, H3K4me3, H3K9me2, and H3K9me3) by western blot, and the impact on and characteristics of spheroid growth when exposed to these drugs. Mouse xenografts were used to evaluate the anticancer effect of sequential drug treatment.Results:Combination treatment had greater efficacy than single drugs and significantly suppressed cell viability, migration, and spheroid formation and growth. Sequential treatment of cisplatin (1 mg kg−1) followed by TSA (0.3 mg kg−1) significantly suppressed tumorigenicity of HEY xenografts through inhibition of EMT and decreased pluripotency of ovarian cancer cells.Conclusion:Epigenetic modifiers potentiate the anticancer efficacy of low-dose cisplatin in ovarian cancer through regulation of EMT and pluripotency, and may provide a promising treatment for ovarian cancer patients.

Abstract 2990: The combination of Trichostatin A and 5-aza-2’-deoxycytidine achieves superior anticancer efficacy on ovarian cancer via regulation of migration, invasion, apoptosis, and cell cycle.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Objective: To investigate the anticancer efficacy and mechanism of Trichostatin A (TSA) and 5-aza-2’-deoxycytidine (5-aza-CdR) on ovarian cancer cells. Material and methods: The effect of TSA and 5-aza-CdR was evaluated on the human ovarian cancer cell line SKOV3 in vitro and in vivo. Optimal dose combinations of TSA and 5-aza-CdR was assessed by MTS assay, migration and invasion by transwell assay and BD invasion chamber, expression of epithelial to mesenchymal transition (EMT) markers (Twist, E-cadherin, and N-cadherin), MMP-2/9, apoptosis markers (Bcl-2 and Bcl-x/L), cell cycle markers (p21 and p27), epigenetic markers (DNMT3A/3B, HDAC1/2, LSD1, Ac-H3/H4 and H3K4me2, H3K9me2) by Western blot. Spheroid formation was evaluated in serum free medium when exposed to the combination of drugs. Tumorigenicity of cancer cells treated both before and after implantation subcutaneously and intraperitoneally was assessed in SCID mice. Results: The effect of fixed ratio combinations of TSA and 5-aza-CdR on cell viability was assessed and 0.1 μM TSA and 5 μM 5-aza-CdR achieved the maximal synergistic effect (Combination Index =0.421). Tumorigenicity of SKOV3 cells pretreated with both drugs was significantly suppressed when compared with either drug alone or untreated control (p<0.001) in SCID mice models. Migration capacity was markedly suppressed through the induction of E-cadherin and suppression of N-cadherin (MET), and invasion was suppressed at least partially via inhibition of MMP-2 and MMP-9 with the combined treatment. The combination drugs markedly inhibited both spheroid size (p<0.001) and spheroid number (p=0.002), through inhibition of Twist, N-cadherin, MMP-2, MMP-9 and induction of E-cadherin. The combination of TSA (0.1 mg/kg) and 5-aza-CdR (5 mg/kg) significantly suppressed tumor implantation (p<0.001), and no apparent toxicity was found (control vs. combined, mice weight, p=0.0537) and expression of Bcl-2 and Bcl-x/L was suppressed while p21 and p27 were induced in tumor nodules. Epigenetic markers DNMT3A/3B and HDAC1/2, enzymes important to tumor suppressor genes, were markedly inhibited with the combination. Acetylation of histone H3 and H4 were more markedly stimulated with the combination than with either agent alone. H3K4me2 expression was induced and H3K9me3 and LSD1 were suppressed by exposure to the combined drugs. Conclusion: These results suggest that the combination of TSA and 5-aza-CdR significantly suppresse tumorigenicity of ovarian cancer cells by inhibiting migration and invasion via suppression of N-cadherin and MMP-2/9, induction of E-cadherin, regulation of apoptosis and cell cycle via suppression of Bcl-2 and Bcl-x/L and induction of p21 and p27, which may be epigenetically regulated by DNA methylation and histone modification. Citation Format: Molly A. Brewer, Fanliang Meng, Guigin Sun, Yanhong Yu. The combination of Trichostatin A and 5-aza-2’-deoxycytidine achieves superior anticancer efficacy on ovarian cancer via regulation of migration, invasion, apoptosis, and cell cycle. [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 2990. doi:10.1158/1538-7445.AM2013-2990

Abstract 3953: The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids.

Objective: Recent evidence suggests that the human omentum functions as a niche that promotes ovarian cancer metastasis. We investigated the role of the omentum in sequestration and enhancement of ovarian cancer stem cells. Materials and Methods: To further explore this unique phenomenon, we examined the ability of cells isolated from ovarian cancer implants at distinct anatomic sites to create multicellular aggregates known as spheroids. In ovarian and other cancers, spheroids promote a pluripotent phenotype and play a critical role in metastasis. Results: We found that ovarian cancer cells recovered from omentum express higher levels of the pluripotency-associated antigen CD133 and create larger numbers of more rapidly growing spheroids than cells recovered from the tubo-ovarian complex. Although CD133 + or CD44 + ovarian cancer cells are equally capable of creating large spheroids, spheroids self-assembled from CD133 + ovarian cancer cells are significantly more tumorigenic in vivo than CD44 + , CD133 − CD44 − or unsorted cells. We also found that spheroids self-assembled from CD133 + ovarian cancer cells migrate to both the ovary and omentum of SCID mice and that CD133+ ovarian cancer cells induce intraperitoneal xenografts when inoculated subcutaneously. In contrast, our data indicate that spheroids self-assembled from CD44 + cells preferentially induce tumor adjacent to their inoculation site. Targeting CD133 expression inhibits the in vivo tumorigencity of spheroids assembled from A2780 ovarian cancer cells. Conclusions: In addition to delineating novel pathways by which the omental microenvironment promotes ovarian cancer metastasis, these findings establish a new paradigm for understanding how ovarian cancer stem cells can be potentially maintained or regenerated within the peritoneal cavity. Strategies to target the omental niche have enormous potential to improve ovarian cancer outcomes. Citation Format: Molly A. Brewer, Guigin Sun, Matthew Anderson, Fanliang Meng, Mei Zhong. The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids. [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 3953. doi:10.1158/1538-7445.AM2013-3953

Abstract 1041: Epigenetic regulation of BRAF-HDAC complex in presence of trichostatin A, 5-aza-2′-deoxycytidine and cisplatin in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: To investigate the regulation of the subunits of BRAF-HDAC complex, a gene transcription repression complex, which regulates the interaction of histone methylation, histone acetylation, DNA methylation and DNA repair in response to chemotherapy in ovarian cancer cell lines with different treatment strategies. We hypothesized that combining different epigenetic modifiers with low dose Cisplatin would result in an optimal therapeutic regime for ovarian cancer. Materials and Methods: Two epigenetic modifiers Trichostatin A (TSA), a histone deacetylase inhibitor and 5-aza-2′- deoxycytidine (Decitabine), a demethylating agent were evaluated in different doses alone and in combination with Cisplatin on the expression of subunits of BRAF-HDAC complex, histone methylation, acetylation and the associated pathways affected. Three cell lines, A2780, Hey and SKOV3 were used. Spheroids were grown in serum free medium and injected IP in SCID mice. Western blots confirmed protein expression. Results: Different combinations of TSA, Decitabine and low dose Cisplatin significantly inhibited the expression of the subunits LSD1 and CoREST in the BRAF-HDAC complex, two proteins which together demethylate H3K4 and H3K9. The BRAF-HDAC complex cofactor subunit BRAF35 was also significantly suppressed by the combination treatment. The BRAF-HDAC complex subunit repressor increased significantly after combined treatment. indicating that the combination of TSA, Decitabine and Cisplatin suppresses the expression of H3K4 and H3K9 methylation and increases the expression of Histone H3 lys9/14 and Histone H4 ser1/lys5/lys8/lys12 acetylation. Along with these methylation and acetylation changes, we found a decrease in epithelial-mesenchymal transition, cancer cell proliferation, chemoresistance and pluripotency. Spheroid formation was decreased in vitro and there was no tumor formation in SCID mice in presence of these epigenetic modifiers and low dose Cisplatin. Conclusions: The combination of epigenetic modifiers and low dose Cisplatin can regulate and modify the expression of subunits of BRAF-HDAC complex, resulting in decreased expression of tri- and di- methylated H3K4 and H3K9 and increased acetylation of Histone H3 and H4, subsequently modifying the tumor biology and tumorigenicity of these ovarian cancer cell lines. Further investigation of these mechanisms could provide the development of more effective and less toxic treatment for ovarian cancer. 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 1041. doi:1538-7445.AM2012-1041

Abstract 1406: A new ovarian cancer metastasis model using multicellular spheroids generated from human ovarian cancers tissues

Objective Most research aimed at dissecting the mechanism by which epithelial ovarian cancer (EOC) metastasizes has focused on the ability of individual cells to attach and create tumor implants. However, multicellular aggregates known as spheroids are frequently found in ovarian cancer ascites and these aggregates are increasingly thought to play an important role in metastasis not only for ovarian cancer but also other solid tumors where similar phenomena have been observed. We investigated characteristics of spheroids compared to cells grown in monolayer to further characterize these spheroids. Methods IRB approval was obtained for collection of human tissue. Fresh specimens of ovarian cancer were minced, enzymatically digested, rinsed and incubated either in DMEM to generate monolayer cultures or Mammosphere media to generate spheroids. Tumorigenicity was assessed by inoculating individual spheroids or an equal number of dissociated cells into the peritoneal cavity of SCID mice. Gene expression was examined using Western blot and quantitative real-time PCR. Results We found that cells from most ovarian cancers do generate spheroids and most of the spheroids generated are able to reliably induce peritoneal implants in SCID mice. At a molecular level, the spheroids were enriched for expression of specific markers including CD133, CD44, NANOG and OCT4, associated with populations of cancer initiating cells (CICs) as well as other stem cell markers. MiR-26b, characteristic of CICs, was significantly up regulated in spheroids compared to monolayer cells. Spheroids from CD133 positive cells induced ovarian and abdominal tumors in SCID mice, however, CD44 spheroids only generated localized tumor, suggesting that CD133 is important in metastasis of ovarian cancer. Conclusion Our results indicate that populations of cells from human EOC are capable of spontaneously generating multicellular aggregates that resemble the spheroids found in ovarian cancer ascites de novo. These spheroids are enriched for expression of markers including CD133, CD44, NANOG and OCT4, suggesting that spheroid formation enhances stem cell-like markers. CD133 might also serve as a necessary marker for metastasis of ovarian cancer. The increased expression of miR-26b in spheroids compared to monolayer culture is a novel finding that needs further investigation. 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 1406. doi:1538-7445.AM2012-1406

Abstract 3517: Epigenetic interactive regulation and synergistic action of Trichostatin A, 5-aza-2′-deoxycytidine and Cisplatin in ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective: To investigate the relationship between histone modification, DNA methylation and DNA damage repair response that occur in platinum resistant ovarian cancer cell lines treated with epigenetic modifiers in combination with low dose chemotherapy to develop new approaches for chemotherapy resistant ovarian cancer. Materials and Methods: We selected two common epigenetic modifiers Trichostatin A (TSA), a histone deacetylase inhibitor and 5-aza-2′-deoxycytidine (decitabine), a demethylating agent which were evaluated alone and in combination with low dose Cisplatin for their tumorigencity in SCID mice and their spheroid formation in serum-free medium with two ovarian cancer cell lines SKOV3 and Hey. We also measured the effect of these drugs on tumor suppression pathways, AKT pathway, and expression of cadherins as well as histone modification. Results: All combinations of TSA, decitabine and Cisplatin inhibited both tumorigenicity and spheroid formation in both cell lines with Hey exhibiting more resistance to treatment. E-cadherin expression was suppressed in SKOV3 cells while N-caderin expression was suppressed in Hey cells with both TSA and Cisplatin and decitabine and Cisplatin. In the Hey cell line, p53 expression was increased by sequential treatment with low dose decitabine and low dose cisplatin and by low dose TSA and low dose cisplatin. The profiling of histone code revealed distinctly different chromatin regulation between Hey cells and SKOV3 cells. The expression patterns and characteristics of cell adhesion, mutiple-drug transporter, apoptosis are distinctly different in these two ovarian cancer cell lines, which may be due to the different origins and stages of these cell lines. Conclusions: The combinations of decitabine, TSA and Cisplatin suppressed the formation and growth of xenograft and tumor spheroid, altered the molecular mechanism of cell adhesion, chemosensitivity, and chromatin modification which may provide a new and promising treatment strategy for chemoresistant ovarian cancer. 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 3517. doi:10.1158/1538-7445.AM2011-3517

Abstract 4312: Identification of putative stem cell markers of CD133 and CD44 in human ovarian cancer specimens and ovarian cancer cell lines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objective: Ovarian cancer may arise from a subpopulation of CD133+ 44+ cells, which are self-renewing tumor-initiating cells. Materials and Methods: We investigated the tumorigenicity of the cell line A2780 which is CD133high44low, the cell line A2780 with siRNA against CD133 (CD133low44low), and human ovarian cancer cells from ascites, from primary ovarian cancer, and from metastatic ovarian cancer (CD133 high/CD44high) harvested from patients at the time of surgical cytoreduction in ultra low attachment plates in serum free medium. Spheroids from each of these sources were used to test their tumorigenicity in SCID mice. Results: Cells from A2780 cell line (CD133 high/CD44low) formed tumors in SCID mice in 2 months but silencing CD133 with siRNA CD133 (CD133 low/CD44low) inhibited tumorigenicity in SCID mice and retarded but did not prevent spheroid formation. Spheroid cultures of ascites, primary ovary, and metastasis (CD133 high/CD44high) are capable of inducing primary ovarian tumors and metastatic tumors in SCID mice in 2 months compared to 4-6 months in sorted, non-spheroid cells. Conclusions: The data suggest that CD133 expression is necessary for tumorigenesis in the A2780 cell line but not for spheroid formation. Our data also suggests that spheroids which are CD133 high/CD44high from human tumor recapitulate ovarian cancer in women. These novel in vivo and vitro models may offer useful tools to develop therapies targeted to 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 4312.

Abstract 4231: Epigenetic regulation and tumorigenicity of ovarian cancer cells

Objective: The objective of this study was to investigate changes in CD133 expression and in tumorigenesis that occur in ovarian cancer cell lines treated with drugs that alter gene methylation. Materials and Methods: We selected two common drugs 5-aza-2′-deoxycytidine, a demethylating agent and Trichostain A(TSA), a histone deacetylase inhibitor and investigated three ovarian cancer cell lines(CD133+ cell lines A2780 and CD133- cell lines Hey and SKOV3) to determine their effect on ovarian cells in vitro and in vivo. Results: We demonstrated that epigenetic modification did not significantly alter cell viability but suppressed spheroid formation of cancer cells lines. FACS results indicated that the percentage of CD133+ cells increased following treatment with 5uM 5-aza-2′-deoxycytidine and 300nM trichostatin A after 72° incubation. Xenograft assay of the NOD/SCIDmice results demonstrated that both drugs reduced spheroid formation and blocked tumorigenesis. Conclusions: We concluded that 5-aza-2′-deoxycytidine and TSA can significantly suppress both spheroid formation and tumor formation and could be provide a new strategy for the treatment of resistant 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 4231.