Search

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy, largely due to metastasis and drug resistant recurrences. Fifteen percent of ovarian tumors carry mutations in BRCA1 or BRCA2, rendering them vulnerable to treatment with PARP inhibitors such as olaparib. Recent studies have shown that TGFβ can induce “BRCAness” in BRCA wild-type cancer cells. Given that TGFβ is a known driver of epithelial to mesenchymal transition (EMT), and the con-nection between EMT and metastatic spread in EOC and other cancers, we asked if TGFβ and EMT alter susceptibility of EOC to PARP inhibition. Epithelial EOC cells were transiently treated with soluble TGFβ and their clonogenic potential, expression and function of EMT and DNA repair genes, and response to PARP inhibitors compared with untreated controls. A second epithelial cell line was compared to its mesenchymal derivative for EMT and DNA repair gene expression and drug responses. We found that TGFβ and EMT resulted in downregulation of genes responsible for homologous recombination (HR) and sensitized cells to olaparib. HR efficiency was reduced in a dose-dependent manner. Furthermore, mesenchymal cells displayed sensitivity to olaparib, cis-platin, and the DNA-PK inhibitor Nu-7441. Therefore, treatment of disseminated, mesenchymal tumors may represent an opportunity to expand clinical utility of PARP inhibitors and similar agents.

The effects of Î"b-Twist1 overexpression on spheroids formation and migration. (A) Representative images of spheroid forming assay. (B) Representative images of the migrated cells in migration assay. (C) Quantification of the migrated cells.*P< 0.05 compared to control group.

Supplementary Tables

The protein levels of Twist1 (A) and p53 (B) in tumor samples. Protein levels were determined by the quantification of western blot band intensity comparing to GAPDH bands as loading control.

Expression of p53-associated E3 ligase in cancer cell lines. (A) RNA expression of MDM2, Pirh2, and E6-AP1, and TOPORS assessed by QPCR in R182 (p53high/Twist-) and Tara R182 (p53low/Twist+) cells. GADPH was used as reference gene. (B) Protein expression of Pirh2 assessed by Western blot. β-actin was used as loading control.

TP53 mutation status and protein levels of p53 and Twist1 ovarian cancer cell lines. * u = unknown.

Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully re-acquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally non-functional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a sub-classification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and has adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterize the IFNβ-mediated anti-viral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication and determine whether components of the IFN-induced response can be used as a replacement therapy for ZIKA virus infection during pregnancy. We identify and characterize interferon-stimulated gene 20 (ISG20) as playing a central role in controlling Zika virus infection in trophoblast cells and successfully establish a recombinant ISG20-Fc protein that effectively decreases viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying potential immune modulatory functions. Recombinant ISG20-Fc has thus the potential to be further developed as an anti-viral treatment against ZIKA viral infection in high-risk populations, particularly in pregnant women.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

Epithelial–mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype. Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and leads to adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterized the IFNβ-mediated antiviral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication; and determined whether we could use the components of the IFN-induced response as a replacement therapy for ZIKA viral infection during pregnancy. We identified and characterized interferon stimulated gene 20 (ISG20), playing a central role in controlling Zika infection in trophoblast cells, and successfully established a recombinant ISG20-Fc protein that effectively decrease viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying immune modulatory functions. Therefore, rISG20-Fc is a promising anti-viral/immune modulatory approach for the control and prevention of RNA viral infections such as ZIKA virus.

Epithelial ovarian cancer is the deadliest gynecologic cancer, due in large part to recurrent tumors. Recurrences tend to have metastasized, mainly in the peritoneal cavity and developed resistance to the first line chemotherapy. Key to the progression and ultimate lethality of ovarian cancer is the existence of extensive intra-tumoral heterogeneity (ITH). In this review, we describe the genetic and epigenetic changes that have been reported to give rise to different cell populations in ovarian cancer. We also describe at length the contributions made to heterogeneity by both linear and parallel models of clonal evolution and the existence of cancer stem cells. We dissect the key biological signals from the tumor microenvironment, both directly from other cell types in the vicinity and soluble or circulating factors. Finally, we discuss the impact of tumor heterogeneity on the choice of therapeutic approaches in the clinic. Variability in ovarian tumors remains a major barrier to effective therapy, but by leveraging future research into tumor heterogeneity, we may be able to overcome this barrier and provide more effective, personalized therapy to patients.

Introduction: Ovarian cancer metastasizes via the transcoelomic route and yields overwhelmingly numerous micrometastases. This clinical presentation limits the value of standard of care and contributes to patient mortality. Whereas EMT is required in the acquisition of a migratory phenotype, MET has been shown to occur on secondary sites leading to the establishment of metastasis. The nature of the ovarian cancer cells that result after MET is, however, yet to be fully characterized. Whether these secondary epithelial ovarian cancer cells (sEOC) are required to repeat EMT in order to generate additional metastatic sites is not known. The objectives of this study are twofold: to determine if full MET occurs on secondary sites, and to determine mechanisms that confer to disseminated ovarian cancer cells the ability to rapidly spread and form the characteristic micrometastatic disease. Materials and Methods: Ovarian cancer spheroids were derived from three epithelial ovarian cancer (EOC) cell lines (OVCAR3, R182, R2615). sEOC were derived from the spheroids by plating in tissue culture-treated plates. Gene signature pattern was determined by RNAseq. mRNA and protein levels were quantified by RT-qPCR and Western blot, respectively. TWIST-1 activity was measured by a luciferase reporter system. Promoter binding was determined by anti-CBX7 chromatin IP followed by PCR. CBX7 was knocked out (KO) using CRISPR-Cas9. Tumorigenic potential was quantified by measuring resulting intraperitoneal burden (g) in nude mice. Results: PCA and clustering analysis of RNA-seq data showed sEOC do not fully recapitulate the molecular signature of the primary EOC cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintain a high expression of the classical mesenchymal effector, TWIST-1. TWIST-1 is, however, transcriptionally inactive in these cells as it is inhibited by the PcG protein, CBX7. KO of CBX7 in sEOC was sufficient to promote a mesenchymal morphology, significantly promote TWIST-1-induced transcription of mir-199A (p = 0.004, wt vs. CBX7KO), and induce higher protein levels of FOXC2. CBX7 precludes TWIST-1 from accessing its promoter by binding at or near the E box. Finally, KO of CBX7 was able to significantly enhance tumorigenicity and increase tumor load and metastatic potential in vivo (p=0.033, wt vs CBX7 KO). Conclusion: We demonstrate that full MET does not occur in ovarian cancer cells at secondary metastatic sites. Although sEOC display an epithelial morphology, they do not achieve the full molecular signature of the primary EOC cells from which they are derived. sEOC maintain high levels of TWIST-1 whose activity is inhibited by CBX7. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage to achieve a prompt reversal to a mesenchymal morphology and gain in tumorigenic potential upon perturbation of CBX7. This may explain ovarian cancer’s rapid progression and characteristic micrometastatic disease. Citation Format: Juanni Li, Ayesha B Alvero, Sudhakar Nuti, Roslyn Tedja, Cai M. Roberts, Mary Pitruzzello, Yimin Li, Qing Xiao, Sai Zhang, Yaqi Gan, Gil Mor, Gang Yin. CBX7 binds TWIST-1’s E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A02.

A growing body of evidence has demonstrated the promising anti-tumor effects of resveratrol in ovarian cancer cells, including its inhibitory effects on STAT3 activation. Nonetheless, the low bioavailability of resveratrol has reduced its attractiveness as a potential anti-cancer treatment. In contrast, pterostilbene, a stilbenoid and resveratrol analog, has demonstrated superior bioavailability, while possessing significant antitumor activity in multiple solid tumors. In this study, the therapeutic potential of pterostilbene was evaluated in ovarian cancer cells. Pterostilbene reduces cell viability in several different ovarian cancer cell lines by suppressing cell cycle progression and inducing apoptosis. Further molecular study has shown that pterostilbene effectively suppressed phosphorylation of STAT3, as well as STAT3 downstream genes that regulate cell cycle and apoptosis, indicating that inhibition of STAT3 pathway may be involved in its anti-tumor activity. The addition of pterostilbene to the commonly used chemotherapy cisplatin demonstrated synergistic antiproliferative activity in several ovarian cancer cell lines. Pterostilbene additionally inhibited cell migration in multiple ovarian cancer cell lines. The above results suggest that pterostilbene facilitates significant anti-tumor activity in ovarian cancer via anti-proliferative and pro-apoptotic mechanisms, possibly via downregulation of JAK/STAT3 pathway. Pterostilbene thus presents as an attractive non-toxic alternative for potential adjuvant or maintenance chemotherapy in ovarian cancer.

Breast cancer is the 2nd leading cause of cancer related deaths among women in the US with over 240,000 diagnoses and 40,000 deaths expected in 2014. Among the more serious and deadly forms of breast cancer are the Triple Negative Breast Cancers (TNBC) (ER-, PR-, HER2-). Mortality rates among patients rise dramatically when these cancers spread beyond the primary tumor site. Therefore reduction of tumor cell dispersion is a key component to minimizing mortality rates. Epithelial-Mesenchymal Transition (EMT) is the process by which cancer cells downregulate proteins associated with cell to cell adhesion (e.g. E-cadherin) resulting in cells that are able to detach from neighboring cancer cells, invade adjacent tissue, and eventually enter the circulatory system or lymphatics. The process of EMT is tightly regulated by the transcription factor Twist1, which is often overexpressed in breast cancer. Therefore, Twist1 serves as an excellent therapeutic target whose downregulation would result in fewer metastatic cancer cells and correspondingly reduce cancer mortality. Our lab has designed and tested (in vitro) two siRNA based therapeutics that target Twist1 in a TNBC cell line (SUM 1315). These siRNA molecules have been strategically designed and modified to make them resistant to degradation, enhance silencing effects and diminish their immunogenicity. To overcome the inherent problems with delivery of siRNA (both in vitro and in vivo) we have been testing two nanoparticle delivery systems. Recent advances in nanotechnology have led to the development of a variety of nanoparticles that provide valuable tools for cancer therapy. We are testing two different types of nanoparticles in this study: The first is a Polyamidoamine (PAMAM) dendrimer (YTZ3-15) which is a truncated 3rd generation dendrimer which has been modified with a lipophilic tail to enhance cellular uptake. The second is a mesoporous silica nanoparticle (MSNs) which is able to not only deliver siRNA, but is also contains pores which allow simultaneously delivering chemotherapies such as doxorubicin. This novel siRNA gene silencing and nanotechnology-based therapy should allow us to exert more precise temporal control during breast cancer treatment. When complexed with either nanoparticle we have found significant Twist1 knock down in vitro as well as reduction of Twist1 target genes that are associated with EMT. We have also shown that Twist1 silencing reduces migration and invasion of SUM 1315 breast cancer cells in vitro Recently we began testing these siRNA+Nanoparticle complexes in an orthotopic murine model using Firefly Luciferase expressing SUM 1315 cells in immunocompromised mice (NSG). The results of this in vivo research have shown that there is significant uptake of the siRNA+Nanoparticle complexes in the tumors when compared to other organs. Results of these studies are significant because TNBC is particularly drug resistant and metastatic; and superior therapies are urgently needed to effectively treat patients with these breast cancers. This approach paves the way for TNBC treatment that incorporates Twist1 knock down (resulting in renewed chemosensitivity) and simultaneous delivery of a chemotherapy reagent. Citation Format: James B Finlay, Cai M Roberts, Gina Lowe, Ling Peng, Jeff I Zink, Fuyuhiko Tamanoi, Carlotta A Glackin. Treatment of metastatic breast cancer using two nanoparticles combined with siRNA targeting Twist1 to inhibit EMT [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-04.

Endometrial cancer is the most common gynecologic cancer in the United States and its incidence and mortality has been rising over the past decade. Few treatment options are available for patients with advanced and recurring endometrial cancers. Novel therapies, which are frequently toxic, are difficult to establish in this patient population which tends to be older and plagued by comorbidities such as diabetes mellitus and hypertension. Therefore, novel, non-toxic therapies are urgently needed. Megestrol acetate is a frequently used drug in endometrial cancer patients. However, its response rate is only 20–30%. To enhance the activity of megestrol acetate in endometrial cancer patients, we explored the potential of combining natural supplements with megestrol acetate and found that the addition of the natural phenolic compound, pterostilbene, to megestrol acetate resulted in a synergistic inhibition of cancer cell growth in vitro and an enhanced reduction of tumor growth in a xenograft mouse model. In addition, dual treatment led to attenuation of signaling pathways, as well as cell cycle and survival pathways. Our results demonstrated for the first time that the anti-tumor activity of megestrol acetate can be enhanced by combining with pterostilbene, providing an insight into the potential application of pterostilbene and megestrol acetate combination for the treatment of endometrial cancer.

Epithelial ovarian cancer (EOC) is the most deadly gynecologic malignancy on account of its late stage at diagnosis and frequency of drug resistant recurrences. Novel therapies to overcome these barriers are urgently needed. TWIST is a developmental transcription factor reactivated in cancers and linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance, making it a promising therapeutic target. In this work, we demonstrate the efficacy of TWIST siRNA (siTWIST) and two nanoparticle delivery platforms to reverse chemoresistance in EOC models. Polyamidoamine dendrimers and mesoporous silica nanoparticles (MSNs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with cisplatin plus MSN-siTWIST exhibited lower tumor burden than mice treated with cisplatin alone, with most of the effect coming from reduction in disseminated tumors. This platform has potential application for overcoming the clinical challenges of metastasis and chemoresistance in EOC and other TWIST overexpressing cancers.

Background Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance. Methods In this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion. Results We found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment. Conclusions These data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3169-9) contains supplementary material, which is available to authorized users.

PURPOSE: Metastasis and carcinomatosis remains a major problem in ovarian cancer. Metastasis requires a process known as Epithelial-to-Mesenchymal Transition (EMT), where by epithelial cancer cells undergo dynamic changes to acquire a mesenchymal and invasive phenotype. Twist1 is a transcription factor with a central role in EMT. We previously showed that in epithelial ovarian cancer (EOC), Twist1 is constitutively ubiquitinated and targeted for proteasomal degradation and inhibition of Twist degradation is associated with mesenchymal phenotype and chemoresistance. Accordingly, identification of mechanisms that promote EMT by inhibiting the active degradation of Twist1 is critical for prevention of carcinomatosis. In this study, we report the identification of PKCα as a central regulator of Twist1. We identified 11 possible PKCα phosphorylation sites on Twist1. We show that Twist1 is a novel substrate of the kinase PKCα and that PKCα-induced Twist1 phosphorylation abrogates Twist1 ubiquitination leading to its stabilization and consequently EMT. METHODS: The following cell lines were used: (1) ovarian cancer cells lines OVCAR3 and OVCA432; (2) in-house developed cultures of ovarian cancer cells, R182 and R2615; and (3) HEK293T cells. The effect of constitutively active (PKCαCAT), dominant negative (PKCαDN), and wild-type (wt-PKCα) PKCα on levels of Twist1 were determined by transient transfection using qRT-PCT and western blot analysis. Levels of phosphorylated Twist1 were measured in Twist1 immunoprecipitate-complex using anti-phospho-serine/threonine/tyrosine antibody. The phospho-deficient mutant Twist1 S144A was constructed using QuickChange Site-directed Mutagenesis Kit. PKCα was knocked-out in ovarian cancer cells using CRISPR/Cas9. EMT was induced by treatment with 10 ng/ml TGFβ1 and confirmed molecularly (i.e. loss of epithelial markers E-cadherin, Ck18, Claudin 3 and gain of mesenchymal markers Twist1, N-cadherin, and vimentin). Intra-peritoneal tumors were established by injecting 10 million ovarian cancer cells in athymic nude mice. RESULTS: Transfection with PKCαCAT(active) resulted in a significant increase in Twist1 protein levels compared to PKCαDN(inactive) or empty vector control in EOC and HEK293T cells. This was not a transcriptional effect (no increase in Twist1 mRNA) but due to increase in the levels of phospho-serine/threonine/tyrosine on Twist1. We identified S144 as the putative PKCα phosphorylation site on Twist1 protein. Whereas wt-Twist1 was readily ubiquitinated when transfected in ovarian cancer cells and HEK293T, the phosphomimic S144D Twist1 demonstrated significantly less ubiquitination. Furthermore, we identified TGFβ1 as an activator of the endogenous PKCα-Twist1 axis in ovarian cancer cells. TGFβ1 is able to: (1) activate PKCα; (2) increase Twist1 protein levels; (3) and induce EMT (spheroid formation). Knock-out of PKCα in ovarian cancer cells abrogated TGFβ1-induced EMT in vitro and inhibit carcinomatosis in vivo in athymic nude mice model. CONCLUSION: We demonstrate for the first time a TGFβ1-PKCα-Twist1 signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization. This is a non-classical pathway of TGFβ1 induced EMT. Moreover, we identify S144 on Twist1 as novel and direct PKCα-phosphorylation site that can control Twist1 stability. Given the pleiotropic nature of TGFβ1 signaling, the identification of PKCα as a novel target may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation in ovarian cancer. Citation Format: Roslyn Tedja, Cai Roberts, Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Yang Yang-Hartwich, Carlotta Glackin and Gil G. Mor. PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-084.

Epithelial ovarian cancer (EOC) is the most deadly gynaecologic malignancy due to late onset of symptoms and propensity towards drug resistance. Epithelial-mesenchymal transition (EMT) has been linked to the development of chemoresistance in other cancers, yet little is known regarding its role in EOC. In this study, we sought to determine the role of the transcription factor TWIST1, a master regulator of EMT, on cisplatin resistance in an EOC model. We created two Ovcar8-derived cell lines that differed only in their TWIST1 expression. TWIST1 expression led to increased tumour engraftment in mice, as well as cisplatin resistance in vitro. RNA sequencing analysis revealed that TWIST1 expression resulted in upregulation of GAS6 and L1CAM and downregulation of HMGA2. Knockdown studies of these genes demonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin, but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, in part via GAS6 and L1CAM, led to higher expression and activation of Akt upon cisplatin treatment, and inhibition of Akt activation sensitized cells to cisplatin. These results suggest TWIST1- and EMT-driven increase in Akt activation, and thus tumour cell proliferation, as a potential mechanism of drug resistance in EOC.

Homologous recombination among repetitive sequences is an important mode of DNA repair in eukaryotes following acute radiation exposure. We have developed an assay in Saccharomyces cerevisiae that models how multiple DNA double-strand breaks form chromosomal translocations by a nonconservative homologous recombination mechanism, single-strand annealing, and identified the Rad52 paralog, Rad59, as an important factor. We show through genetic and molecular analyses that Rad59 possesses distinct Rad52-dependent and -independent functions, and that Rad59 plays a critical role in the localization of Rad52 to double-strand breaks. Our analysis further suggests that Rad52 and Rad59 act in multiple, sequential processes that determine genome structure following acute exposure to DNA damaging agents.

Growth and progression of solid tumors depend on the integration of multiple pro-growth and survival signals, including the induction of angiogenesis. TWIST1 is a transcription factor whose reactivation in tumors leads to epithelial to mesenchymal transition (EMT), including increased cancer cell stemness, survival, and invasiveness. Additionally, TWIST1 drives angiogenesis via activation of IL-8 and CCL2, independent of VEGF signaling. In this work, results suggest that chemically modified siRNA against TWIST1 reverses EMT both in vitro and in vivo . siRNA delivery with a polyethyleneimine-coated mesoporous silica nanoparticle (MSN) led to reduction of TWIST1 target genes and migratory potential in vitro . In mice bearing xenograft tumors, weekly intravenous injections of the siRNA-nanoparticle complexes resulted in decreased tumor burden together with a loss of CCL2 suggesting a possible anti-angiogenic response. Therapeutic use of TWIST1 siRNA delivered via MSNs has the potential to inhibit tumor growth and progression in many solid tumor types. From the Clinical Editor Tumor progression and metastasis eventually lead to patient mortality in the clinical setting. In other studies, it has been found that TWIST1, a transcription factor, if reactivated in tumors, would lead to downstream events including angiogenesis and result in poor prognosis in cancer patients. In this article, the authors were able to show that when siRNA against TWIST1 was delivered via mesoporous silica nanoparticle, there was tumor reduction in an in-vivo model. The results have opened up a new avenue for further research in this field.

Background: Epithelial - mesenchymal transition (EMT) is a pre-requisite for cancer metastasis. Twist-1 is a transcription factor with a central role in the process of EMT and therefore metastasis formation. Using ovarian cancer models, our group has previously shown that although the stationary epithelial phenotype express Twist1 mRNA, this phenotype is enforced by mechanisms that constitutively ubiquitinates and degrades the Twist1 protein. The objective of this study is to identify molecular mechanisms that promote Twist1 protein stabilization and therefore confer a mesenchymal and mobile phenotype. Materials and methods: Novel phosphorylation sites on Twist1 protein, which are adjacent to ubiquitination sites, were identified using NetPhosK and Ubpred. Further analysis demonstrates that most of these sites contain the consensus sequence for PKCα. Constitutively active PKCα (PKCαcat) was ectopically expressed in HEK293T cells and patient-derived epithelial ovarian cancer cell lines. Protein expression and phosphorylation status were determined using western blot analysis using either whole cell lysates or cellular fractions. Protein-protein interaction was determined by immunoprecipiation. The specific Twist1 domain required for PKCα interaction was determined by co-transfection with Twist1 deletion mutants. Results: Ectopic expression of PKCαcat in HEK293T cells and ovarian cancer cells resulted in increased levels of Twist1 protein compared to empty vector control. In ovarian cancer cells, PKCcat-induced upregulation of Twist1 protein is not associated with an increase in Twist1 mRNA but instead is associated with Twist1 phosphorylation, decreased ubiquitination, and enhanced stabilization. We identify the twist box (WR) domain of Twist1 as a pre-requisite for PKCα binding and PKCαcat-induced Twist1 stabilization. Moreover, we identify TGFβ1 as a potent activator of endogenous PKCα in epithelial ovarian cancer cells. TGFβ1 (1 ng/ml) is able to: promote the activation and membranal translocation of PKCα; increase Twist1 protein levels; and induce spheroid formation and EMT in epithelial ovarian cancer cells. Interestingly, TGFβ1 had no effect on Twist1 mRNA suggesting that this mechanism is independent of the classical TGFβ-Smad pathway. Conclusions: We demonstrate for the first time a novel TGFβ-PKCα signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization resulting in EMT. This mechanism is independent of the classical TGFβ-Smad pathway that controls EMT via transcriptional regulation. Since TGFβ is a pleiotropic cytokine that can affect multiple cell types, the identification of PKCα as a novel target in ovarian cancer cells may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation. Citation Format: Roslyn Tedja, Ayesha B. Alvero, Carlos Cardenas, Mary Pitruzzello, Gang Yin, Yang Yang-Hartwich, Cai Roberts, Carlotta Glackin, Gil G. Mor. PKCα-induced Twist1 phosphorylation is a novel regulator of Twist1 stabilization [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 1969. doi:10.1158/1538-7445.AM2017-1969

Breast cancer is the leading cause of cancer-related deaths among women in the United States, and survival rates are lower for patients with metastases and/or triple-negative breast cancer (TNBC; ER, PR, and Her2 negative). Understanding the mechanisms of cancer metastasis is therefore crucial to identify new therapeutic targets and develop novel treatments to improve patient outcomes. A potential target is the TWIST1 transcription factor, which is often overexpressed in aggressive breast cancers and is a master regulator of cellular migration through epithelial-mesenchymal transition (EMT). Here, we demonstrate an siRNA-based TWIST1 silencing approach with delivery using a modified poly(amidoamine) (PAMAM) dendrimer. Our results demonstrate that SUM1315 TNBC cells efficiently take up PAMAM-siRNA complexes, leading to significant knockdown of TWIST1 and EMT-related target genes. Knockdown lasts up to one week after transfection and leads to a reduction in migration and invasion, as determined by wound healing and transwell assays. Furthermore, we demonstrate that PAMAM dendrimers can deliver siRNA to xenograft orthotopic tumors and siRNA remains in the tumor for at least four hours after treatment. These results suggest that further development of dendrimer-based delivery of siRNA for TWIST1 silencing may lead to a valuable adjunctive therapy for patients with TNBC.

Breast cancer in women is a leading cause of cancer related deaths in the United States with metastatic disease being the chief cause of mortality. Metastatic breast cancer tumors are often found in their later stages making them difficult to remove or treat. Inhibition of tumor cell dispersion is therefore a key component to a reduction in mortality rates among women with breast cancer. Epithelial-Mesenchymal Transition (EMT) is the process by which cancer cells downregulate proteins associated with cell to cell adhesion (e.g. E-cadherin) resulting in cells that are able to mobilize. Breast cancer cells are known to commandeer the EMT process through overexpression of Twist1, thus allowing the cells to metastasize beyond the primary tumor. Twist1 therefore serves as an excellent therapeutic target whose disruption would result in fewer metastatic cancer cells and correspondingly reduce cancer mortality. Twist1 is also a desirable target because it is almost nonexistent in adult tissues and thus its silencing would have minimal side effects. Our lab has elected to use an siRNA mediated gene silencing approach to decrease the expression levels of Twist1 in two highly invasive breast cancer cell lines (SUM 1315 and MCF7-Tw). Due to their fragile nature, siRNA molecules are often difficult to deliver at therapeutic levels. We have overcome these delivery limitations through the optimization and use of Poly (amidoamine) (PAMAM) dendrimers. We hypothesize that suppression of the activity of Twist1 via dendrimer-delivered Twist1 siRNA will inhibit metastatic behavior of aggressive breast cancer cells. Here we demonstrate successful delivery of Cy3-labled siRNA using two different dendrimers (Generation 5 and a modified Generation 3) with transfection efficiency results exceeding those of Lipofectamine 2000 and with far less toxicity to cells. We also show up to a 90% reduction (lasting at least 4 days) in Twist1 expression using Western Blot and qPCR analysis. Phenotypically we were able to appreciate a significant decrease in the invasive nature of the breast cancer cells using migration/invasion assays. Taken together these results demonstrate successful knockdown of Twist1 using siRNA dendrimer complexes resulting in an altered cellular phenotype. These data will serve as a foundation for optimization of future in vivo experiments with both orthotopic and metastatic breast cancer models. Citation Format: James B. Finlay, Cai M. Roberts, Carlotta A. Glackin. Dendrimer delivery of RNA based Twist1 inhibitors to reduce breast cancer cell metastasis. [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 2692. doi:10.1158/1538-7445.AM2013-2692