Trevor G. Shepherd

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasivess and resistance to anti-invasive action of TGFβ

Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)β. Since EVT cells possess active invasion‐associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12–15 passages, SV40 Tag‐transformed cells were selected on the basis of extended lifespan. A long‐lived line, RSVT‐2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation‐induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation‐regulating growth factor TGFβ and changes in gene expression for invasion‐associated enzymes or enzyme inhibitors. RSVT‐2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti‐proliferative and anti‐invasive signals from TGFβ. Since both cell lines remained non‐tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)‐1, while TIMP‐2 and plasminogen activator inhibitor (PAI)‐1 expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti‐invasive action of TGFβ was explained by the failure of TGFβ to upregulate TIMPs and PAI‐1, in contrast to the TGFβ‐induced upregulation noted in parental HTR8 cells. Int. J. Cancer 77:429–439, 1998.© 1998 Wiley‐Liss, Inc.

Modulation of AKT activity is associated with reversible dormancy in ascites-derived epithelial ovarian cancer spheroids

Epithelial ovarian cancer (EOC) metastasis is a direct contributor to high recurrence and low survival for patients with this disease. Metastasis in EOC occurs by cell exfoliation from the primary tumor into the fluid-filled peritoneal cavity, persistence of these cells as non-adherent multicellular aggregates or spheroids and reattachment of spheroids to form secondary lesions. We have recovered native spheroids from ascites fluid and demonstrated that EOC cells within these structures exhibit reduced proliferation, yet regain the capacity to attach and reinitiate cell division. To model this process in vitro for further investigation, primary EOC cells from patient peritoneal fluid were cultured under non-adherent conditions. Here we show that these cells naturally form spheroids resembling those observed in ascites. Spheroids exhibit reduced cell proliferation and a protein expression pattern consistent with cellular quiescence: specifically, decreased phospho-AKT and p45/SKP2 with a concomitant increase in p130/RBL2 and p27(Kip1). However, when spheroids are seeded to an adherent surface, reattachment occurs rapidly and is followed by reinitiation of AKT-dependent cell proliferation. These results were strikingly consistent among numerous clinical specimens and were corroborated in the EOC cell line OVCAR3. Therefore, our data reveal that EOC cells become quiescent when forming spheroids, but reactivate proliferative mechanisms upon attachment to a permissive substratum. Overall, this work utilizes a novel in vitro model of EOC metastasis that employs primary human EOC cells and introduces the important concept of reversible dormancy in EOC pathogenesis.

Contribution of reactive oxygen species to ovarian cancer cell growth arrest and killing by the anti-malarial drug artesunate.

Ovarian cancer is a leading cause of cancer‐related death in women and the most lethal gynecological malignancy in the developed world. The morbidity and mortality of ovarian cancer underscore the need for novel treatment options. Artesunate (ART) is a well‐tolerated anti‐malarial drug that also has anti‐cancer activity. In this study, we show that ART inhibited the in vitro growth of a panel of ovarian cancer cell lines, as well as the growth of ovarian cancer cells isolated from patients. Moreover, ART decreased tumor growth in vivo in a mouse model of ovarian cancer. ART‐treated ovarian cancer cells showed a strong induction of reactive oxygen species (ROS) and reduced proliferation. ROS‐dependent cell cycle arrest occurred in the G2/M phase whereas ROS‐independent cell cycle arrest occurred in the G1 phase, depending on the concentration of ART to which ovarian cancer cells were exposed. The anti‐proliferative effect of ART was associated with altered expression of several key cell cycle regulatory proteins, including cyclin D3, E2F‐1, and p21, as well as inhibition of mechanistic target of rapamycin signaling. Exposure of ovarian cancer cells to higher concentrations of ART resulted in ROS‐dependent DNA damage and cell death. Pretreatment of ovarian cancer cells with a pan‐caspase inhibitor or ferroptosis inhibitor decreased but did not completely eliminate ART‐mediated cytotoxicity, suggesting the involvement of both caspase‐dependent and caspase‐independent pathways of killing. These data show that ART has potent anti‐proliferative and cytotoxic effects on ovarian cancer cells, and may therefore be useful in the treatment of ovarian cancer.

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non‐tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti‐cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2‐targeting second‐generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non‐tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti‐cancer agent.

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability.

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence.

Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity

OBJECTIVE We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV). METHODS Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus protein expression, and cell viability by alamarBlue assay. Akti-1/2 (5 μM) and rapamycin (20 nM) were used to assay the role of PI3K-AKT signaling in mediating MYXV infection. RESULTS Ascites-derived EOC cells grown in adherent culture are effectively killed by MYXV infection. EOC cells grown in suspension to form three-dimensional EOC spheroids readily permit MYXV entry into cells, yet are protected from the cytopathic effects of late MYXV infection. Upon reattachment (to model secondary metastasis), EOC spheroids are re-sensitized to MYXV-mediated oncolysis. The critical determinant that facilitates efficient MYXV infection is the presence of an activated PI3K-AKT signaling pathway. Treatment with the specific AKT inhibitor Akti-1/2 reduces infection of monolayer EOC cells and spheroids. Direct infection of freshly-collected ascites demonstrated that 54.5% of patient samples were sensitive to MYXV-mediated oncolytic cell killing. We also demonstrate that factor(s) present in ascites may negatively impact MYXV infection and oncolysis of EOC cells, which may be due to a down-regulation in endogenous AKT activity. CONCLUSIONS Differential activity of AKT serves as the mechanistic basis for regulating MYXV-mediated oncolysis of EOC spheroids during key steps of the metastatic program. In addition, we provide the first evidence that MYXV oncolytic therapy may be efficacious for a significant proportion of ovarian cancer patients with metastatic disease.

TGFβ signaling regulates epithelial–mesenchymal plasticity in ovarian cancer ascites-derived spheroids

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease.

BMP signalling controls the malignant potential of ascites-derived human epithelial ovarian cancer spheroids via AKT kinase activation

Epithelial ovarian cancer (EOC) cells have the ability to form multi-cellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Herein, we demonstrate that patient ascites-derived EOC cells down-regulate endogenous bone morphogenetic protein (BMP) signalling by decreasing BMP ligand expression when grown in suspension culture to form spheroids. Enforced BMP signalling in these cells via constitutively-active BMP type I ALK3QD receptor expression causes the formation of smaller, more loosely-aggregated spheroids. Additionally, ALK3QD-expressing spheroids have an increased rate of adhesion and dispersion upon reattachment to substratum. Inhibition of endogenous BMP signalling using recombinant Noggin or small molecule inhibitor LDN-193189, on the other hand, opposed these phenotypic changes. To identify potential targets that impact the phenotype of EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. Using the online Connectivity Map resource, the BMP signalling gene expression signature revealed that the AKT pathway is induced by activated BMP signalling in EOC cells; this finding was further validated by phospho-AKT immuno-blotting. In fact, treatment of EOC spheroids with an AKT inhibitor, Akti-1/2, reduced BMP-stimulated cell dispersion during reattachment as compared to controls. Thus, we have identified AKT as being one important downstream component of activated BMP signalling on EOC spheroid pathobiology, which may have important implications on the metastatic potential of this malignancy.

Evidence for Differential Viral Oncolytic Efficacy in an In Vitro Model of Epithelial Ovarian Cancer Metastasis

Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis.

The Human Papillomavirus E7 Proteins Associate with p190RhoGAP and Alter Its Function

ABSTRACT Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.