<p> <u>Background and Rationale:</u> Although the majority of patients with endometrial cancer (EC) are diagnosed early when disease is confined in the uterus and prognosis is excellent, there is a subset of patients with dismal prognosis. Carboplatin and paclitaxel is the standard chemotherapeutic regimen for EC. Given that response to chemotherapy impacts disease prognosis, especially in advanced, recurrent and metastatic disease, novel chemotherapeutic agents with improved safety profile are necessary to improve response rates and outcomes in these patients. Quinacrine (QC) is an inexpensive antimalarial drug with a predictable safety profile which recently surfaced as a promising anticancer agent thought to be associated with decreased risk of developing chemo-resistance through targeting multiple pathways simultaneously. </p><p> <u>Objective:</u> To generate preclinical data on the effect of QC in inhibiting tumorigenesis in EC both <i>in vitro</i> and <i>in vivo</i> as well as explore its role as an adjunct to standard chemotherapy <i>in vivo</i> in an EC mouse xenograft model. </p><p> <u>Methods:</u> Five different EC cell lines (Ishikawa, Hec-1B, KLE, ARK-2, and SPEC-2) representing different histologies, grades of EC, sensitivity to cisplatin and p53 status were used for the <i> in vitro</i> studies. MTT and colony formation assays were used to examine QC&rsquo;s ability to inhibit cell viability <i>in vitro.</i> Drug combination studies were performed and the Chou-Talalay methodology was employed in order to examine synergism between QC and cisplatin, carboplatin or paclitaxel. A cisplatin-resistant EC subcutaneous mouse xenograft model was used in order to explore QC&rsquo;s anticancer activity <i>in vivo</i> and assess its role as maintenance therapy. </p><p> <u>Results:</u> QC exhibited strong synergism <i> in vitro</i> when combined with cisplatin, carboplatin or paclitaxel with the highest level of the synergism being observed in the most chemo-resistant EC cell line. Neither QC monotherapy nor standard chemotherapy significantly delayed tumor growth in the mouse xenografts. Co-administration of QC with standard chemotherapy significantly augmented the antiproliferative ability of these chemotherapeutic agents as evidenced by the significant decrease in tumor burden. Combination treatment was associated with a 14-week prolongation of median survival compared to standard chemotherapy alone. Maintenance therapy with QC following standard chemotherapy was proven superior to standard chemotherapy as it resulted in long-term stabilization of disease evidenced by lack of significant tumor progression and further prolongation of overall survival. QC treatment alone, in combination with standard chemotherapy or as maintenance therapy was well-tolerated and was not associated with weight loss compared to control mice. A yellow skin discoloration was noted during active treatment with QC which was entirely reversible within a few days upon discontinuation of treatment. </p><p> <u>Conclusions:</u> QC exhibited significant antitumor activity against EC cell lines <i>in vitro</i> and was successful as maintenance therapy in chemo-resistant EC mouse xenografts. This preclinical data suggest that QC may be an important adjunct to standard platinum-based chemotherapeutic regimens for patients with recurrent EC.</p>

<p> <i>Sushi Domain Containing 2 (SUSD2)</i> encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. A clinically annotated HGSOC tissue microarray was stained with an anti-SUSD2 antibody. Patients with tumors that had weak SUSD2 staining had a shorter median survival (31.7 months) compared to patients that had tumors with strong SUSD2 staining (49.1 months; p value = 0.0083).</p><p> To investigate the role of SUSD2 in HGSOC, stable OVCAR3, OVSAHO and KURAMOCHI cell lines were established through transfection of shRNA targeted to <i>SUSD2</i> transcripts (SUSD2 knock-down [KD] cell lines) or non-targeting shRNA (SUSD2 NT) cell lines. Boyden chamber and wound healing assays demonstrated that OVCAR3, OVSAHO and KURAMOCHI SUSD2 KD cells migrated at significantly higher rates than their SUSD2 NT counterpart cell lines. RT-qPCR and western immunoblot analysis indicated an inverse relationship between SUSD2 and well characterized mesenchymal genes, such as <i>TWIST1, ZEB1</i> and <i>CHD2.</i> In addition, OVCAR3 and KURAMOCHI SUSD2 KD spheroids displayed increased mesothelial clearance ability compared to SUSD2 NT spheroids.</p><p> To explore the potential for SUSD2 to inhibit late-stage HGSOC metastasis, female athymic nude mice were injected with either OVCAR3 NT or OVCAR3 KD cells. Fewer nodules were observed in the pancreas and omentum of the OVCAR3 NT mice when compared to the OVCAR3 KD mice. Furthermore, OVCAR3 KD mice had a significantly shorter median survival compared to OVCAR3 NT mice (175 days compared to 185.5 days, respectively; p-value = 0.0047).</p><p> KURAMOCHI lysate was immunoprecipitated for SUSD2-associated immunocomplexes and subjected to liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis, yielding a list of candidate SUSD2-interacting proteins associated with RNA processing. Immunofluorescence analysis of OVCAR3, KURAMOCHI and SKBR3 cells and western immunoblot analysis of their subcellular extracts revealed SUSD2 to be present in cell nuclei, mitochondria and cytoplasm; however, SUSD2 was relatively less abundant in SKBR3 nuclei.</p><p> Our findings suggest that increased <i>SUSD2</i> expression in HGSOC impedes metastasis, consistent with prolonged survival observed in HGSOC patients with high <i>SUSD2</i>-expressing primary tumors. The differences in subcellular distribution between HGSOC cells and breast cancer cells may explain alternate functions of SUSD2 in different cancers.</p><p>

Oncolytic adenoviral gene therapy has potential as a novel anti-cancer agent for ovarian cancer. Host immune responses are thought to contribute to its therapeutic effects. However further evaluation has been hampered by the lack of an immunocompetent animal model. This is predominantly because human adenovirus is highly species-specific and replicates poorly in murine cells. The second generation human adenovirus (hAd5) type 5 mutant dl922-947 contains a deletion in the E1A CR2 region which allows it to replicate selectively in cells with Rb pathway abnormalities, a finding observed in >90% of human cancers. Previous work has shown that dl922-947 has considerable activity in ovarian cancer and is more potent than E1A wild-type adenoviruses and the E1B-55K mutant dl1520 (Onyx-015, H101). Unfortunately, like its wild-type counterpart, dl922-947 replicates poorly in murine cells and infectious virion progeny are not generated. Mechanisms for the failure of infectious virion formation remain unclear and have been investigated as part of this project. I have found that murine malignant cells can be infected readily with hAd5 vectors. Both early and late viral genes are transcribed and there is evidence of viral genome replication. However, a profound failure of infective virion production is observed together with low levels of late viral protein expression. Ribosome fractionation assays show reduced viral mRNA loading in murine cells, resulting in failure of translation, especially of late transcripts. Aberrant function of the non-structural L4 protein 100K has been identified as a major hurdle to successful viral replication in murine cells. Ectopic expression of L4 100K promotes translation of viral late mRNA and increases expression of late viral proteins and virion production. However, these increases are only partial.