Mary Pitruzzello

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.

Glucose and metformin modulate human first trimester trophoblast function: a model and potential therapy for diabetes-associated uteroplacental insufficiency.

Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin.

TRX-E-002-1 Induces c-Jun–Dependent Apoptosis in Ovarian Cancer Stem Cells and Prevents Recurrence In Vivo

Chemoresistance is a major hurdle in the management of patients with epithelial ovarian cancer and is responsible for its high mortality. Studies have shown that chemoresistance is due to the presence of a subgroup of cancer cells with stemness properties and a high capacity for tumor repair. We have developed a library of super-benzopyran analogues to generate potent compounds that can induce cell death in chemoresistant cancer stem cells. TRX-E-002-1 is identified as the most potent analogue and can induce cell death in all chemoresistant CD44+/MyD88+ ovarian cancer stem cells tested (IC50 = 50 nmol/L). TRX-E-002-1 is also potent against spheroid cultures formed from cancer stem cells, chemosensitive CD44−/MyD88− ovarian cancer cells, and heterogeneous cultures of ovarian cancer cells. Cell death was associated with the phosphorylation and increased levels of c-Jun and induction of caspases. In vivo, TRX-E-002-1 given as daily intraperitoneal monotherapy at 100 mg/kg significantly decreased intraperitoneal tumor burden compared with vehicle control. When given in combination with cisplatin, animals receiving the combination of cisplatin and TRX-E-002-1 showed decreased tumor burden compared with each monotherapy. Finally, TRX-E-002-1 given as maintenance treatment after paclitaxel significantly delayed disease recurrence. Our results suggest that TRX-E-002-1 may fill the current need for better therapeutic options in the control and management of recurrent ovarian cancer and may help improve patient survival. Mol Cancer Ther; 15(6); 1279–90. ©2016 AACR.

Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation.

Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1β production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1β response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1β processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.

Adipocyte microenvironment promotes Bclxl expression and confers chemoresistance in ovarian cancer cells.

Resistance to mitochondria-initiated apoptosis is a hallmark of chemoresistant cancer stem cells including CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells. This is controlled by members of the Bcl2 family of proteins, which function as rheostats of mitochondrial stability. We observed a differential expression profile of Bcl2 family members comparing the chemoresistant EOC stem cells and the chemosensitive CD44−/MyD88− EOC cells. Chemoresistant EOC stem cells surprisingly express higher levels of the pro-apoptotic members Bak and Bax compared to the chemosensitive EOC cells. In addition, whereas chemosensitive EOC cells preferentially express Bcl2, chemoresistant EOC stem cells preferentially express Bclxl. In the EOC stem cells, 40% knock-down of Bclxl expression was sufficient to induce the full activation of caspases and this can be reversed by concurrent knock-down of Puma. More importantly, we demonstrate that Bclxl expression levels in EOC cells is dynamic and can be regulated by microenvironments that are enriched with the pro-inflammatory cytokine IL-6 such as the cancer stem cell and adipocyte niches. Adipocyte-induced upregulation of Bclxl correlated with acquisition of chemoresistance and thus demonstrates how a specific microenvironment can regulate the expression of apoptotic proteins and confer chemoresistance.

Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model

Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still relies primarily on white-light reflectance and the surgeon’s vision. As such, micrometastases are usually missed and most patients clinically classified as a complete responder eventually recur and succumb to the disease. Our objective is to develop optical enhancers which can aid in the visualization of ovarian cancer micrometastasis. To this end we developed a nanoparticle (NP) platform, which is specifically targeted to the tumor microenvironment. Targeting is achieved by coating FDA-approved PLGA-PEG NP with the peptide sequence RGD, which binds with high affinity to αVβ3 integrins present in both the tumor-associated neovasculature and on the surface of ovarian cancer cells. Administration of the NP platform carrying fluorescent dyes to mice bearing intraperitoneal ovarian cancer allowed visualization of tumor-associated vasculature and its contrast against normal blood vessels. More importantly, we demonstrate the visualization of intraperitoneal ovarian cancer micrometastasis as small as 100 μm with optimal resolution. Finally, we demonstrate that the fluorescent dye cargo was able to penetrate intra-tumorally. Such modality could be used to allow microscopic surgical debulking to assure maximal surgical effort.

Abstract 1995: The omentum promotes ovarian cancer cell survival by increasing cell cycle duration and chemoresistance

Background: Ovarian cancer is the most lethal of all gynecologic cancers. It is characterized by the presence of carcinomatosis at time of presentation and chemoresistant micrometastatic seedings at the time of recurrence. The omentum, an adipocyte-rich organ, is a frequent site of ovarian cancer metastasis in patients and the most common site of post-chemotherapy residual disease in animal models. Whereas previous studies have shown how the adipocyte microenvironment can induce metabolic re-programming in cancer cells, it is not clear if it can regulate pathways that affect chemoresponse. The objective of this study is to determine the effect of the adipocyte microenvironment on pathways that control cell cycle and apoptosis. Methods: In vitro: Patient-derived epithelial ovarian cancer cell lines were cultured in the presence of absence of omentum conditioned-media. Cell growth was determined by measuring culture confluence using IncucyteTM. Protein expression was determined by western blot analysis, and flow cytometry. In vivo: Human ovarian cancer xenografts were established intra-peritoneally in nude mice. Tumor implants from adipocyte-rich organs such as the omentum, pelvic fat, and mesentery and tumor implants from non-adipocyte-rich organs such as liver, ovaries, and GI tract were collected and analyzed independently. Results: Compared to cells cultured in growth media, epithelial ovarian cancer cells cultured in omentum CM demonstrated a more fibroblastic morphology characterized by elongated shape and bipolarity, significantly slower growth kinetics (p 50 μg/ml. Analysis of tumor implants in vivo showed similar results as observed in vitro. Thus, tumor implants isolated from adipocyte-rich organs express higher levels of p27, Bclxl, and Mcl-1. Conclusion: We demonstrate in this study that the adipocyte microenvironment induces major changes in the phenotype of ovarian cancer cells characterized by morphological changes chemoresistance and growth rate. These findings highlight the importance of the adipocyte microenvironment in the progression of ovarian cancer. Further studies that can identify specific therapeutic targets in the adipocyte-educated chemoresistant ovarian cancer cells may aid in the development of novel therapies and improve patient survival. Citation Format: Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Roslyn Tedja, Gil G. Mor. The omentum promotes ovarian cancer cell survival by increasing cell cycle duration and chemoresistance [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 1995. doi:10.1158/1538-7445.AM2017-1995

Abstract A62: TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model.

Background: Ovarian cancer is the leading cause of mortality from gynecologic cancers due to the high incidence of chemoresistance and disease recurrence. Disease recurrence is thought to occur due to the presence of residual disease following first-line standard of care (i.e. optimal debulking and chemotherapy). Residual disease post chemotherapy is composed of a unique population of chemoresistant cancer cells with stemness properties and a high capacity for tumor repair. Currently, there are no available options that can target these cells. The objective of this study was to develop a new therapeutic modality that will target chemoresistant ovarian cancer stem cells (OCSC) and consequently prevent recurrence and improve survival. To achieve this objective we developed a library of super-benzopyran (SBP) analogues and identified TRX-1 as the most potent analogue able to induce OCSC cell death in a short period of time. Moreover, in contrast to Cisplatin and Paclitaxel, TRX-1 is able to induce a persistent growth inhibitory effect both in vitro and in vivo thus improving overall survival in a mouse model of recurrent ovarian cancer. Materials and methods: A panel of SBP analogues were generated and activity was determined by testing against pure clones of CD44+/MyD88+ OCSC. In vitro efficacy was assessed using the IncucyteTM kinetic imaging platform complemented by CelltoxTM dye labeling. In vivo efficacy was tested using an intra-peritoneal (i.p.) a mouse model of recurrent ovarian cancer1. Results: TRX-1 was the most potent analogue identified and is able to induce cell death in all OCSC clones tested (IC50 of 136 nM). We observed that 2h in vitro exposure to 2μM TRX-1 was sufficient to induce a sustained growth inhibitory effect in OCSC, hence the cells were not able to recover growth potential even after removal of the drug. In contrast, OCSC exposed for up to 24h with the same dose of Cisplatin or Paclitaxel were able to recover. In vivo, animals bearing residual tumors after Paclitaxel treatment demonstrated tumor progression when further maintained with vehicle or Paclitaxel indicating Paclitaxel resistance. In contrast, maintenance with TRX-1 was able to effectively decrease tumor burden (p = 0.02) and prevent recurrence. In addition, combination treatment with TRX-1 and Cisplatin was able to significantly improve survival (p Conclusion: We describe the in vitro and in vivo anti-tumoral effect of a novel compound, TRX-1, which exhibits significant efficacy against chemoresistant OCSC and is able to prevent recurrence in a chemoresistant in vivo model. Recurrence characterized by chemoresistance is the main cause of mortality in ovarian cancer patients. Previous studies from our laboratory have shown that conventional chemotherapy is not effective against OCSC and cannot prevent recurrence. Our finding that TRX-1, by targeting OCSC can prevent recurrence in vivo as maintenance therapy or in combination with chemotherapy provides a new opportunity for developing new therapeutic strategies that can improve survival in ovarian cancer patients. Citation Format: Ayesha Alvero, Eydis Lima, Mary Pitruzzello, Yang Yang-Hartwich, David Brown, Andrew Heaton, Gil Mor. TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A62.

Abstract A61: Detection of ovarian cancer micrometastasis using nanoparticle-delivered probe targeted towards tumor-associated neovasculature.

Background: Ovarian cancer causes more death than all other gynecologic malignancies combined. In patients, the best predictor of overall survival is the amount of residual disease following surgical debulking. Indeed, studies have shown that patients have the best prognosis when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still primarily relies on white-light reflectance and the surgeon9s vision. As such, micrometastasis are usually missed and most patients clinically classified as a complete responder carry residual tumor burden, eventually recur, and succumb to the disease. We hypothesize that we can utilize the specific phenotype of tumor-associated neovasculature to target optical enhancers to locate and delineate micrometastasis. Specific targeting is achieved by encapsulating deep infrared dye (DIR) in FDA-approved PLGA-based nanoparticle (NP) coated with the peptide sequence arginine-glycine-aspartate (RGD). RGD binds with high affinity to αVβ3 integrins, which are minimally expressed in quiescent blood vessels and normal cells but overexpressed in tumor-associated neovasculature and cancer cells of various origins, including ovarian cancer. Thus our objective is to develop specific tumor-targeting optical enhancers that can aid surgeons in the performance of microscopic tumor debulking with the goal of minimizing residual disease. Materials and methods: Intra- peritoneal (i.p.) tumors are established in nude mice using ovarian cancer cells that stably express mCherry fluorescent protein, which allows the colocalization of DIR signal and mCherry+ micrometastatic lesions. Using this model we compared the ability of soluble DIR, DIR encapsulated in naked NP (DIR-NP), and DIR encapsulated in RGD-coated NP (DIR-RGD-NP) to detect ovarian cancer micrometastasis. Formulations were delivered i.p. and fluorescence signal were measured ex vivo following necropsy. More importantly, the ability of the probe to locate and delineate micrometastatic lesions was determined using fluorescent dissection microscope. Results: Colocalization analysis of DIR and mCherry fluorescent images obtained at necropsy demonstrated the sensitivity of DIR-RGD-NP. In these animals, we observed 75% colocalization of DIR and mCherry signals compared to 26% in animals administered DIR-NP and 0% in those given soluble DIR. Ex vivo analysis of mean DIR intensity in tumors less than 2 mm showed mean fluorescent intensity (MFI) of 1209 in animals administered DIR-RGD-NP versus 155 MFI in animals administered DIR-NP. Morphological examination under the dissection microscope show that in animals administered DIR-RGD-NP, we can locate 81% of the mCherry+ micrometastasis based on DIR staining. In these animals, tumors less than 1 mm were detectable due to a halo of DIR staining around each micrometastatic lesion. In these foci, DIR signal was observed to stain the vasculature surrounding the small tumor implants especially those in the mesentery and diaphragm. In contrast, we were able to detect only 18% of the mCherry+ micrometastasis in animals administered DIR-NP. Conclusion: We demonstrate that we can visualize micrometastasis by using RGD as a tool to target the unique phenotype of ovarian cancer-associated neovasculature. RGD-coated nanoparticles are able to carry probes to the tumor microenvironment leading to optimal staining of micrometastasis. Our results highlight the use of this nanotechnology platform in microscopic surgical debulking to assure maximal surgical effort, minimize residual disease, and improve patient survival. Furthermore, delineation of tumor borders will also reduce inadvertent injury to vital structures and decrease surgery-associated morbidities. Citation Format: Ayesha Alvero, Eydis Lima, Dongin Kim, Sean Orton, Mary Pitruzzello, Yang Yang-Hartwich, Dan-Arin Silasi. Detection of ovarian cancer micrometastasis using nanoparticle-delivered probe targeted towards tumor-associated neovasculature. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A61.

Abstract 4201: Targeting tumor-associated neovasculature for delivery of optical enhancers detects ovarian cancer micrometastasis

Background: Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. While identification and removal of large metastases do not pose a challenge, micrometastases are impossible to distinguish intra-operatively and contribute to the high mortality. Our objective is to develop specific tumor-targeting optical enhancers that can aid surgeons in the performance of microscopic tumor debulking with the goal of minimizing microscopic residual disease. We hypothesize that we can utilize overexpressed αVβ3 integrins in the tumor-associated neovasculature. Specific targeting is achieved by encapsulating fluorescent probes in FDA-approved PLGA-based nanoparticle (NP) coated with the peptide sequence arginine-glycine-aspartate (RGD), which binds with high affinity to these integrins. Materials and methods: Ovarian cancer xenograft is established intra- peritoneally (i.p.) in nude mice using cancer cells that stably express mCherry fluorescent protein. The following formulations were tested: soluble deep infrared dye (DIR), DIR encapsulated in naked NP (DIR-NP), and DIR encapsulated in RGD-coated NP (DIR-RGD-NP). Formulations were delivered i.p. and colocalization of fluorescent signals were determined ex vivo. Staining of micrometastasis was visualized using fluorescent dissection microscope. Results: The best colocalization was observed in mice administered DIR-RGD-NP. We observed 75% colocalization in this group compared to 26% and 0% in DIR-NP and DIR groups, respectively. Ex vivo analysis of DIR intensity in tumors less than 2 mm showed mean fluorescent intensity (MFI) of 1209 in DIR-RGD-NP group versus 155 MFI DIR-NP group. Based on DIR staining, we can locate 81% of mCherry+ micrometastasis in animals administered DIR-RGD-NP. In these animals, tumors less than 1 mm were detected due to a halo of DIR staining around each micrometastatic lesion. In these foci, DIR signal was observed to stain the vasculature surrounding the small tumor implants especially those in the mesentery and diaphragm. In contrast, we were able to detect only 18% of the mCherry+ micrometastasis in animals administered DIR-NP. Conclusion: We demonstrate that we can utilize the specific phenotype of tumor-associated neovasculature to target optical enhancers to locate and delineate micrometastasis. RGD-coated nanoparticles are able to carry probes to the tumor microenvironment leading to optimal staining of micrometastasis. Our results highlight the use of this nanotechnology platform in microscopic surgical debulking to assure maximal surgical effort, minimize residual disease, and improve patient survival. Citation Format: Ayesha B. Alvero, Eydis Lima, Dongin Kim, Sean Orton, Natalia Sumi, Mary Pitruzzello, Yang Yang-Hartwich, Dan-Arin Silasi, Tarek Fahmy, Gil Mor. Targeting tumor-associated neovasculature for delivery of optical enhancers detects ovarian cancer micrometastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4201.