Shake It Up Baby Now: The Changing Focus on TWIST1 and Epithelial to Mesenchymal Transition in Cancer and Other Diseases.

TWIST1 is a transcription factor that is necessary for healthy neural crest migration, mesoderm development, and gastrulation. It functions as a key regulator of epithelial-to-mesenchymal transition (EMT), a process by which cells lose their polarity and gain the ability to migrate. EMT is often reactivated in cancers, where it is strongly associated with tumor cell invasion and metastasis. Early work on TWIST1 in adult tissues focused on its transcriptional targets and how EMT gave rise to metastatic cells. In recent years, the roles of TWIST1 and other EMT factors in cancer have expanded greatly as our understanding of tumor progression has advanced. TWIST1 and related factors are frequently tied to cancer cell stemness and changes in therapeutic responses and thus are now being viewed as attractive therapeutic targets. In this review, we highlight non-metastatic roles for TWIST1 and related EMT factors in cancer and other disorders, discuss recent findings in the areas of therapeutic resistance and stemness in cancer, and comment on the potential to target EMT for therapy. Further research into EMT will inform novel treatment combinations and strategies for advanced cancers and other diseases.

Transforming Growth Factor Beta and Epithelial to Mesenchymal Transition Alter Homologous Recombination Repair Gene Expression and Sensitize BRCA Wild-Type Ovarian Cancer Cells to Olaparib.

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 TGFß can induce "BRCAness" in BRCA wild-type cancer cells. Given that TGFß is a known driver of epithelial to mesenchymal transition (EMT), and the connection between EMT and metastatic spread in EOC and other cancers, we asked if TGFß and EMT alter the susceptibility of EOC to PARP inhibition. Epithelial EOC cells were transiently treated with soluble TGFß, 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 TGFß and EMT resulted in the 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, cisplatin, and the DNA-PK inhibitor Nu-7441. Therefore, the treatment of disseminated, mesenchymal tumors may represent an opportunity to expand the clinical utility of PARP inhibitors and similar agents.

Placenta-derived interferon-stimulated gene 20 controls ZIKA virus infection.

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 IFNß-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.

Flow Cytometric Analyses of p53-Mediated Cell Cycle Arrest and Apoptosis in Cancer Cells.

Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6:a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.

Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes.

CRISPR/Cas9-based gene editing is a recent advance that allows for the knockout or alteration of target genes within mammalian cells. Many variations of the technique exist, but here we describe two systems of plasmid-based CRISPR gene knockout which together allow for the selective knockout of virtually any gene target. Compared with other CRISPR-based systems, these plasmids have the advantages of delivering all the necessary components in one plasmid, choice of multiple selectable markers, and choice of route of administration into target cells. In addition, potential off-target effects from one system (dependent upon selection of target gene) can be overcome through use of the second system. Strategies for optimizing the knockout process and selection of finished cell lines are also presented.

CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.

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 reacquire 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 nonfunctional 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 subclassification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

The Role of Intra-Tumoral Heterogeneity and Its Clinical Relevance in Epithelial Ovarian Cancer Recurrence and Metastasis.

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.

Protein kinase Cα-mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it.

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.

p53-Pirh2 Complex Promotes Twist1 Degradation and Inhibits EMT.

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.

Pterostilbene Suppresses Ovarian Cancer Growth via Induction of Apoptosis and Blockade of Cell Cycle Progression Involving Inhibition of the STAT3 Pathway.

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.

Pterostilbene, a natural phenolic compound, synergizes the antineoplastic effects of megestrol acetate in endometrial cancer.

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.

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target.

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.

Nanoparticle delivery of siRNA against TWIST to reduce drug resistance and tumor growth in ovarian cancer models.

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.

TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6, L1CAM, and Akt signalling.

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.

Mesoporous silica nanoparticle delivery of chemically modified siRNA against TWIST1 leads to reduced tumor burden.

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.

RNA-based TWIST1 inhibition via dendrimer complex to reduce breast cancer cell metastasis.

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.

Rad59 regulates association of Rad52 with DNA double-strand breaks.

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.