Summary High-grade serous ovarian carcinoma presents significant clinical and therapeutic challenges. Although the traditional model of carcinogenesis has focused on the ovary as a tumor initiation site, recent studies suggest that there may be additional sites of origin outside the ovary, namely the secretory cells of the fallopian tube. Our study demonstrates that high-grade serous tumors can originate in fallopian tubal secretory epithelial cells and also establishes serous tubal intraepithelial carcinoma as the precursor lesion to high-grade serous ovarian and peritoneal carcinomas in animal models targeting the Brca, Tp53, and Pten genes. These findings offer an avenue to address clinically important questions that are critical for cancer prevention and early detection in women carrying BRCA1 and BRCA2 mutations.

Chemoresistance to platinum therapy is a major obstacle that needs to be overcome in the treatment of ovarian cancer patients. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). This study demonstrates that the Notch signaling pathway and Notch3 in particular are critical for the regulation of CSCs and tumor resistance to platinum. We show that Notch3 overexpression in tumor cells results in expansion of CSCs and increased platinum chemoresistance. In contrast, γ-secretase inhibitor (GSI), a Notch pathway inhibitor, depletes CSCs and increases tumor sensitivity to platinum. Similarly, a Notch3 siRNA knockdown increases the response to platinum therapy, further demonstrating that modulation of tumor chemosensitivity by GSI is Notch specific. Most importantly, the cisplatin/GSI combination is the only treatment that effectively eliminates both CSCs and the bulk of tumor cells, indicating that a dual combination targeting both populations is needed for tumor eradication. In addition, we found that the cisplatin/GSI combination therapy has a synergistic cytotoxic effect in Notch-dependent tumor cells by enhancing the DNA-damage response, G 2 /M cell-cycle arrest, and apoptosis. Based on these results, we conclude that targeting the Notch pathway could significantly increase tumor sensitivity to platinum therapy. Our study suggests important clinical applications for targeting Notch as part of novel treatment strategies upon diagnosis of ovarian cancer and at recurrence. Both platinum-resistant and platinum-sensitive relapses may benefit from such an approach as clinical data suggest that all relapses after platinum therapy are increasingly platinum resistant.

Nanoscale drug delivery vehicles have been harnessed extensively as carriers for cancer chemotherapeutics. However, traditional pharmaceutical approaches for nanoformulation have been a challenge with molecules that exhibit incompatible physicochemical properties, such as platinum-based chemotherapeutics. Here we propose a paradigm based on rational design of active molecules that facilitate supramolecular assembly in the nanoscale dimension. Using cisplatin as a template, we describe the synthesis of a unique platinum (II) tethered to a cholesterol backbone via a unique monocarboxylato and O→Pt coordination environment that facilitates nanoparticle assembly with a fixed ratio of phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000]. The nanoparticles formed exhibit lower IC 50 values compared with carboplatin or cisplatin in vitro, and are active in cisplatin-resistant conditions. Additionally, the nanoparticles exhibit significantly enhanced in vivo antitumor efficacy in murine 4T1 breast cancer and in K-Ras LSL/+ /Pten fl/fl ovarian cancer models with decreased systemicand nephro-toxicity. Our results indicate that integrating rational drug design and supramolecular nanochemistry can emerge as a powerful strategy for drug development. Furthermore, given that platinum-based chemotherapeutics form the frontline therapy for a broad range of cancers, the increased efficacy and toxicity profile indicate the constructed nanostructure could translate into a nextgeneration platinum-based agent in the clinics.chemotherapy | nanomedicine

Cisplatin is a first line chemotherapy for most types of cancer. However, its use is dose-limited due to severe nephrotoxicity. Here we report the rational engineering of a novel nanoplatinate inspired by the mechanisms underlying cisplatin bioactivation. We engineered a novel polymer, glucosamine-functionalized polyisobutylene-maleic acid, where platinum (Pt) can be complexed to the monomeric units using a monocarboxylato and an O → Pt coordinate bond. We show that at a unique platinum to polymer ratio, this complex self-assembles into a nanoparticle, which releases cisplatin in a pH-dependent manner. The nanoparticles are rapidly internalized into the endolysosomal compartment of cancer cells, and exhibit an IC50 (4.25 AE 0.16 μM) comparable to that of free cisplatin (3.87 AE 0.37 μM), and superior to carboplatin (14.75 AE 0.38 μM). The nanoparticles exhibited significantly improved antitumor efficacy in terms of tumor growth delay in breast and lung cancers and tumor regression in a K-ras LSL∕þ ∕Pten fl∕fl ovarian cancer model. Furthermore, the nanoparticle treatment resulted in reduced systemic and nephrotoxicity, validated by decreased biodistribution of platinum to the kidney as quantified using inductively coupled plasma spectroscopy. Given the universal need for a better platinate, we anticipate this coupling of nanotechnology and structureactivity relationship to rationally reengineer cisplatin could have a major impact globally in the clinical treatment of cancer.chemotherapy | nanomedicine | cancer

This study evaluates the effects of a spearmint (/-carvone) and wintergreen oil (methyl salicylate) emulsion (CMSE) on age 1 landlocked Atlantic salmon Salmo salar sebago (hereafter salmon). Salmon were immersed in either 257 µl/L CMSE or 75 mg/L tricaine methanesulfonate (MS-222) to induce anesthesia (stage 4), useful for emersion and noninvasive husbandry procedures, and then salmon were recovered in fresh water. Induction was quicker in the CMSE group; however, recovery was quicker in the MS-222 group. A second experiment was conducted in which salmon were immersed in 257 µl/L CMSE for 8.5 min, or 75 mg/L MS-222 for 8.5 min in order to compare electrocardiographs during deeper anesthesia (stage 5) between salmon continuously immersed in CMSE to those continuously immersed in MS-222. Because salmon remained sedated longer after CMSE exposure than after MS-222 exposure, a third group of salmon was immersed in 257 µl/L CMSE for just 2.5 min before undergoing the 6-min electrocardiograph procedure. Anesthesia induction rates, recovery rates, and electrocardiographs of salmon anesthetized with CMSE were comparable to salmon anesthetized with MS-222. Salmon anesthetized with CMSE and then transferred immediately to fresh water had more stable heart rates than salmon anesthetized with either MS-222 or CMSE continuously. Salmon bathed continuously in CMSE showed clinical signs of increasing anesthetic depth including decreasing heart rate, decreasing respiration rate and electrocardiograph abnormalities. The CMSE, with its mint and wintergreen concentrations less than in household products such as chewing gum, toothpaste, and mouthwash, is a potent, rapid-acting immersion fish anesthetic comparable to MS-222 for stages 4 and 5 anesthesia.

Ovarian cancer is the most lethal gynecologic malignancy because the vast majority of cases are detected in late stage, a finding that has thwarted attempts to understand the pathogenesis and cell-of-origin of this disease. The traditional view of epithelial ovarian pathogenesis asserts that all tumor subtypes share a common origin in the ovarian surface epithelium (OSE). There is robust data to support the OSE as the site of origin for many ovarian tumors, including low-grade carcinomas and borderline tumors. However, the pathogenesis of high-grade serous ovarian carcinoma, the most common type of ovarian cancer, continued to defy explanation by the OSE model. More recent studies suggested that the fallopian tube epithelium (FTE), rather than the OSE, may be the site-of-origin for a majority of pelvic serous carcinomas (PSC, defined as ovarian, peritoneal and tubal high grade serous carcinomas). We show here that the FTE can be site of origin for PSC by genetically engineering a mouse model that specifically targets the FT secretory cell with defined genetic alterations that are characteristic of human PSC. These mice develop tubal intraepithelial serous carcinomas, a precursor to PSC, that are morphologically and immunophenotypically similar to the lesions described in human patients. Furthermore, these intraepithelial lesions progress to widespread peritoneal disease that recapitulates the presentation of high-grade PSC in women. The tumors express common serous markers such as P53, αH2A.X, PAX8 and CA-125. Taken together our model is the first fully genetically engineered mouse model that truly recapitulates human serous carcinoma pathogenesis. Our model serves as proof-of-concept that the FTE can be site-of-origin to PSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3292. doi:1538-7445.AM2012-3292

High-grade serous ovarian cancer, the most common and deadly subtype of ovarian tumors, presents significant clinical and therapeutic challenges. Effective tools for early detection are not readily available resulting in late diagnosis of often widely metastatic disease. Although research has traditionally focused on the hypothesis that HGSC arises from the ovarian surface epithelium (OSE) or ovarian inclusion cysts, recent studies from BRCA1 and BRCA2 mutation carriers undergoing prophylactic salpingo-oophorectomies suggest that additional sites of origin exist and a substantial proportion of cases may arise instead from precursor lesions located in the fallopian tubal epithelium (FTE). The latter hypothesis is based on the detection of pre-invasive lesions, namely serous tubal intraepithelial carcinomas (STIC), found in the fallopian tubes (FT) of both women at high-risk for developing serous carcinomas as well as patients with disseminated HGSCs. Recent studies of ovarian and tubal tissue samples from high-risk BRCA1/2 carriers who underwent prophylactic salpingo-oophorectomy procedures to reduce cancer risk revealed early cancer lesions in ~5-15% of cases, with 60-100% of lesions found in the FT fimbria. In addition, pre-invasive STICs, which are defined as in situ cancers with p53 mutations and increased proliferative capacity, are observed in at least 60% of women with HGSC of the ovary and/or peritoneum and similar in situ lesions are not observed in the OSE. Clinical observations support the hypothesis that STICs can originate from secretory epithelial cells of the fallopian tube (FTSECs) and progress to HGSC by rapidly disseminating to involve ovarian and peritoneal surfaces. However, these associations are largely circumstantial and necessitate experimental proof in order to confirm the role of FTSECs and STICs in serous carcinogenesis. The vast implications of unequivocally establishing a potential cell of origin in HGSC for both cancer prevention and early tumor detection prompted us to develop animal models that mimic human pathogenesis. To this end we targeted defined genetic human alterations, such as BRCA1, BRCA2, and P53, within mouse fallopian tubal secretory cells (FTSECs) in order to recapitulate the steps of fallopian tube transformation into ovarian cancer, thereby providing the first experimental evidence for a FTSEC origin of HGSC in BRCA carriers. In order to generate a clinically informative mouse model of HGSC, we targeted the FTSEC using specific lineage markers, which are expressed in normal FTSEC and in nearly all HGSC cases but not in the ovary or OSE. In this study, we describe the first mouse model for de novo high-grade serous carcinoma originating in the clinically relevant site of the fallopian tubal epithelium. This model accurately recapitulates the clinical features of the human disease and establishes that the FTSEC and STIC can be a cell-of-origin and precursor lesion for HGSC, respectively. This model further confirms the transformation continuum from STIC to HGSC and mimics the underlying molecular alterations, along with the histological, immunophenotypic, increased CA125 levels, and genomic patterns that have been associated with human high-grade serous carcinomas. These findings offer a new avenue to address clinically important questions that are critical for cancer prevention and early detection in women carrying BRCA1 or BRCA2 mutations. Since our models closely mirror the human disease, they will be of great use to answer many key questions in HGSC pathogenesis. First, it will be possible to establish specific time points at which pre-invasive lesions become invasive in order to shed light on early stage versus metastatic disease. Secondly, these murine models can also be effectively exploited to study FT transformation, and because we have been able to differentiate between secretory and ciliated cells, our model provides the ability to study the role of hormones, ovulation, and follicular fluid in ovarian carcinogenesis. Early detection of HGSC is difficult but the concept is highly attractive because it can conceivably be achieved at less cost and burden to the patient. If a significant proportion of “ovarian” cancers actually emerge from the distal fallopian tube, a significant shift in both conceptual thinking and preventive strategy will be required. One of the most actively debated questions based on this new model of pathogenesis is whether patients at high-risk for high-grade serous ovarian carcinoma should have only the affected distal portion of the fallopian tube removed, rather than both tubes and ovaries. Can these young BRCA women be offered risk-reduction surgery without undergoing surgical menopause and loss of their fertility? Thus, the first goal of our research has been to produce a valid model for BRCA serous high-grade tumors. The second is to identify unique characteristics of early serous carcinogenesis in the tube that can be exploited for early detection and to examine the causes of the earliest events preceding malignancy, which will create a great opportunity for cancer prevention in high-risk women. It is clear that events leading to the development of STICs play a significant role for the development of serous tumors and resolving their pathogenesis will enable more efficient methods for early detection, tumor imaging, cancer prevention, and identification of populations at risk. This abstract is also presented as Poster B4. Citation Format: Gregory A Wyant, Ruth Perets, Katherine Muto, Barish B. Poole, Jin YH Chen, Anders W Ohman, Sunita R Setlur, Christopher P. Crum, Ronny Drapkin, Daniela M. Dinulescu. Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in BRCA/P53 models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR01.

Combination chemotherapy is the mainstay of cancer regimes. However, additive off target effect of the drugs is still a major drawback of combination therapy. Nanoscale drug delivery systems, through enhanced permeation and retention effect, target the tumour more efficiently while reducing side effects. Combination of Cisplatin, a widely used platinum based chemotherapeutic with PI3 kinase/mTOR inhibitor has shown synergism. In this study, we designed a chimeric nanoparticle of Cisplatin and PI828, a PI3 kinase inhibitor. PI828 was chemically conjugated to cholesterol by a carbamate linkage. Cisplatin was coordinated to a cholesterol-succinic acid derivative via a unique monocarboxylato and OαPt bond. The chimeric nanoparticle was formulated by mixing both the conjugates in definite proportions with phosphatidylcholine and DSPE-PEG. The chimeric naoparticle had a consistent size between 100 to 130 nm determined by dynamic light scattering. The in vitro release kinetics experiment showed slow and sustained release of both the drugs over a time period of 96 hours. In vitro cell proliferation assay expressed cytotoxicity of the chimeric nanoformulation in 4T1, a murine breast cancer cell line and in KRAS upregulated and PTEN suppressed ovarian cancer model cells. We have evaluated the activity of chimeric nanoparticle in a K-Ras(LSL/+)/PTEN(fl/fl) ovarian cancer model, with a luciferase reporter system. The mice treated with chimeric nanoparticle exhibited suppressed tumour growth as well as reduced toxicity as compared to the free drug combination and vehicle treated mice. These results indicate that chimeric nanoformulation can better the outcome of Cisplatin and PI828 combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1951. doi:1538-7445.AM2012-1951