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Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Triple negative breast cancer (TNBC) is among the most aggressive clinical manifestations of breast cancer (BC), and represents a significant clinical challenge to the effective treatment of early metastatic and treatment-refractory disease because of its poor outcomes. Therefore, there exists a need to develop pre-clinical models that retain the characteristics of the original TNBC tumor—e.g., PDX tumor models—to better understand the mechanisms of drug resistance in metastatic TNBC and to effectively evaluate the effects of anti-cancer drugs on patients with this disease. Furthermore, quantitative changes in cellular element signatures, which indicate levels of enzymatic activity, are emerging new biomarkers for cancer in the clinic, but are of yet systematically understudied in tumor samples from breast cancer patients or/and PDX models of these tumors. Due to limitations in current cellular element signature quantitation profiles, there also exists a need to investigate further. Seven samples of advanced BC patient pleural effusion were obtained from Northwestern Memorial Hospital to establish a PDX tumor model in immunodeficient NSG female mice using breast fat pad xenografting and to develop derived 3D spheroid cultures for anti-cancer drug evaluation. To authenticate, STR profiling against with original patient tumor DNA was conducted. Five developed BC PDX tumor models were shown by pathology to have highly heterogeneous characteristics and the metastatic features of the origin patient tumor. Liver and lung metastases were observed in breast fat pad xenografted PDX tumor mice. 3D tumor spheroid cultures were successfully established from original BC pleural effusion or/and PDX tumor cultures. Using the newly developed, highly sensitive, and reliable Wash-Free Inductively Coupled Plasma Mass Spectrometry (WF ICP-MS) method, we evaluated the inorganic phenotypes from samples of breast cancer patient tumor tissue and of the established PDX tumor to quantify the mobile elements (Na, K, and Ca) and less mobile elements (P, Mg, Mn, Fe, Cu, and Zn) simultaneously within the same sample. The data was collected for further analysis of breast cancer inorganic signatures from normal and tumor cells. Our results suggested that BC 3D spheroid cultures and PDX tumor models could serve as models to further study the mechanisms of MBC and serve as promising tools for in vivo and in vitro quick testing and mechanistic studies of novel antitumor drugs respectively. Identifying quantitative elemental profiles is fundamental to understanding the pathologies of various metal-related cancers and thus opens up new opportunities for disease management and therapeutic intervention Citation Format: Wenan Qiang, Haimei Chen, Yi Yang, Andrew Crawford, Demirkan B. Gursel, Jian-Jun Wei, Thomas O’Halloran, Massimo Cristofanilli. Establishment of patient-derived xenograft tumor models from breast cancer patients for quantitative elemental profiles and testing candidate therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-03.

Uterine leiomyosarcoma (LMS) is a rare but deadly disease. Due to poor understanding of the molecular and genetic causes of the disease, the diagnosis of LMS has been based primarily on histology. Nuclear atypia is one of hallmarks in LMS, however, it also occurs in 2 clinically benign variants, including smooth muscle tumors with fumarate hydratase alteration (SMT‐FH) and leiomyoma with bizarre nuclei (LM‐BN). In addition to nuclear atypia, many well recognized biomarkers used for LMS are also frequently overexpressed in LM‐BN, and the histogenesis and molecular natures for LM‐BN and LMS remain largely unknown. To characterize the molecular profiling of LMS, SMT‐FH, and LM‐BN, we performed integrated comprehensive genomic profiling including whole‐genome sequencing (WGS) and RNA sequencing and genomic microarray analyses to assess genome‐wide copy number alterations (CNAs) and immunohistochemistry (IHC) in all 3 tumor types. We found that both LM‐BN and LMS showed genomic instability and harbored extensive CNAs throughout the whole genome. By contrast, the SMT‐FH presented its characteristic 1q43‐44 deletions in all cases tested, with minimal CNAs in the rest of genomic regions. Further analyses revealed that LMS and LM‐BN groups showed similar patterns of CNAs that are tended to cluster together and separated from the SMT‐FH group. The integrated molecular profiling enabled the detection of novel and traditional biomarkers and showed excellent discrimination between LM‐BN and LMS. Our study suggests that LM‐BN, despite having similar nuclear atypia to SMT‐FH, showed similar genomic instability but distinct genomic alterations with its malignant counterpart of LMS. The integrated molecular profiling is of clinical importance in characterizing these rare uterine smooth muscle tumors., Leiomyosarcoma and 2 benign mimics (leiomyoma with FH alteration [SMT‐FH] and leiomyoma with bizarre nuclei [LM‐BN]) have characteristic nuclear atypia and often cause diagnostic challenge. LM‐BN and LMS are highly genomically unstable and harbor extensive CNAs in the genome, whereas SMT‐FH shows simple 1q43‐44 deletions. Integrated molecular profiling enabled the detection of novel and traditional biomarkers and showed excellent discrimination between LM‐BN and LMS.

Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7− cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7–β-catenin–Tp63–GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype.Significance:Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

PURPOSE/OBJECTIVE The objective of this study was to assess the association between pretreatment p53, hypoxia inducible factor 1a (HIF1a), Ki-67, carbonic anhydrase-9 (CA-9), and glucose transporter 1 (GLUT1) expression in locally advanced cervical cancer patients treated definitively with concurrent chemoradiation therapy (CRT) and treatment outcomes including overall survival (OS), progression-free survival (PFS), local-regional control (LC), and distant metastases-free survival (DMFS). PATIENTS AND METHODS Twenty-eight patients treated definitively and consecutively for cervical cancer with CRT had p53, HIF1a, Ki-67, CA-9, and GLUT1 protein expression assessed and scored semiquantitatively by 3 pathologists, blinded to the treatment outcomes. Outcomes were stratified by p53 (H-score

Objective To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). Design This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression in vitro; and exploring vessel formation induced by HMGA2 overexpression both in vitro and in vivo. Setting University research laboratory. Patients None. Interventions None. Main Outcome Measures The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and in vitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression in vitro and in vivo. Results Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth in vitro and formed xenografts with increased blood vessels. Conclusions HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

Background: Low- and middle-income countries are facing a high health burden of cervical cancer (CC). The situation is worsened by a high prevalence of human immunodeficiency virus (HIV). We aim to identify epigenetic signatures to help understand the virus-associated pathways underlying CC development, which is fundamental to developing effective CC screening tools or therapeutic approaches for women living with HIV. Methods: We recruited a total of 365 Nigerian women with mean age of 52 (239 HIV+ and 126 HIV-; 98 without CC, 106 with cervical lesions, and 161 with CC). DNA methylation profiling in cervical tissue samples was performed using Illumina EPIC array covering over 860K methylation sites. The epigenetic signatures were identified among HIV+ women through epigenome-wide association study comparing CC vs. CC-free (Bonferroni adjusted p Results: We identified 46 differentially methylated markers (p-value ranging from 5.7e-8 to 2.4e-19) in HIV+ CC women. The effect sizes among the 46 markers in the paired tumor-normal analysis and the paired lesion-normal analysis were highly correlated with the CC vs. CC-free analysis (r=0.99 and r=0.95, respectively) with smaller magnitudes (1.8 and 4.3 times smaller, respectively). Gene ontology and Reactome pathway enrichment analysis revealed that these 46 markers were enriched in genes involving activation of PI3K/AKT/mTOR signaling network (e.g., RPTOR, HDAC3, MAPKAP1), which plays a crucial role in virus/host crosstalk and virus-induced carcinogenesis. The PI3K/AKT/mTOR signaling cascade suppressors, including gene PRDM8, were silenced by promoter hypermethylation among HIV+ CC women (p=4.3e-12). These epigenetic changes, however, were not observed in CC women without HIV. In an independent dataset, MRS was the lowest in women without CC (2.3±2.1), followed by women with cervical lesions (4.9±1.1), and the highest in CC women (9.6±3.9) (p-trend < 2e-16). The MRS achieved areas under the ROC curve = 0.93 and 0.88 in distinguishing CC and cervical lesions from CC-free among HIV+ women, respectively. Conclusion: HIV may communicate with cervical cells and promote CC through epigenetic activation of PI3K/AKT/mTOR signaling pathway. Our epigenetic signatures may serve as novel biomarkers for CC early detection and treatment for women living with HIV. Citation Format: Yinan Zheng, Olugbenga Silas, Jonah Musa, Yishu Qu, Tao Gao, Kyeezu Kim, Brian Joyce, Jun Wang, Demirkan Gursel, Abdulkareem Fatimah, Godwin Imade, Alani Akanmu, Jian-Jun Wei, Masha Kocherginsky, Kwang-Youn Kim, Firas Wehbe, Chad Achenbach, Rose Anorlu, Melissa Simon, Atiene Sagay, Folasade Ogunsola, Robert Murphy, Lifang Hou. Epigenetic signatures of virus-associated cervical cancer in women living with HIV [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3021.

Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. In countries with high human immunodeficiency virus (HIV) prevalence, such as Nigeria, HIV-associated HCC causes a great health burden due to its early onset, late diagnosis, and poorer prognosis. HIV infection is involved in inflammation of the liver, which may determine an increased risk of hepatocyte neoplastic transformation. Inflammation-related DNA methylation signatures obtained in liquid biopsy, such as circulating cell-free DNA (ccfDNA) extracted from serum/plasma are promising minimally-invasive biomarkers that may inform HCC among people living with HIV. Methods: A total of 289 Nigerian participants with information on ccfDNA and other covariates were included. Participants were classified into three groups by their HCC/HIV status: 1) HCC+/HIV+ (n=28); 2) HCC-/HIV+ (n=185); and 3) HCC+/HIV- (n=76). We constructed ccfDNA methylation inflammation scores using 49 CpGs previously linked to circulating C-reactive protein (CRP) concentrations, and higher scores represented elevated CRP concentrations with lower methylation levels. To compare the ccfDNA methylation inflammation score across the groups, we performed multivariable logistic regression analyses adjusting for age, sex, alcohol consumption, smoking status, body mass index, study sites, and technical variables. Results: Participants with HCC+/HIV+ presented the highest ccfDNA methylation inflammation scores (mean=-0.03, standard deviation [SD] =0.01). Participants with HCC-/HIV+ presented the lowest scores (mean=-0.05, SD=0.01). In the multivariable logistic regressions, one SD increase of inflammation score was associated with 2.5 times higher odds of having HCC among HIV-infected participants (HCC+/HIV+ vs. HCC+/HIV-; OR=2.56, 95% CI=1.39-4.73, P=0.002). No evidence was found for the association between the inflammation score and HIV status among HCC patients (HCC+/HIV+ vs. HCC+/HIV-; OR=1.00, 95% CI=0.59-1.70, P=0.991). In the secondary analysis comparing HCC+ vs. HCC- adjusting for HIV status, one SD increase of inflammation score was associated with 3.2 times higher odds of having HCC (OR=3.22, 95% CI=1.39-7.46, P=0.006). Conclusions: We observed that ccfDNA methylation inflammation score is associated with HCC status among people with HIV. Our findings suggest that ccfDNA methylation-based inflammatory profiles may serve as a potential biomarker for early detection and risk stratification of HCC in resource-constrained countries with a high prevalence of HIV. Citation Format: Kyeezu Kim, Yinan Zheng, Claudia Hawkins, Edith Okeke, Olufunmilayo Lesi, Yishu Qu, Lewis R. Roberts, Demirkan Gursel, Fatimah B. Abdulkareem, Alani Akanmu, Godwin Imade, Jian-Jun Wei, Masha Kocherginsky, Kwang-Youn Kim, Wasiu L. Adeyemo, Firas Wehbe, Chad J. Achenbach, Atiene Sagay, Folasade T. Ogunsola, Robert L. Murphy, Lifang Hou. DNA methylation-based inflammation score is associated with hepatocellular carcinoma among people living with HIV. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6013.

Objectives Nuclear factor kappa B (NFkB) is a transcription factor shown to confer treatment resistance in tumors. A previous report suggested an association between pretreatment NFkB and poorer outcomes for cervical cancer patients treated with chemoradiation therapy (CRT). We aimed to validate their findings in a larger patient cohort. Materials and methods This Institutional Review Board approved study included patients with locally advanced cervical cancer patients treated with CRT. Evaluation of both nuclear and cytoplasmic immunoreactivity for NFkB was scored semiquantitatively by 3 pathologists. Cytoplasmic positivity incorporated both the intensity and percentage of immunoreactivity in invasive carcinoma (H-score), whereas nuclear positivity was assessed by percentage of positive cells. Outcomes were stratified by NFkB overexpression and tumor characteristics. Overall survival (OS), progression-free survival (PFS), distant metastases-free survival (DMFS), and local regional control (LC) were obtained using Kaplan-Meier and differences between groups were evaluated by the log-rank test. Hazard ratios were obtained using Cox regression for both univariate and multivariate analyses. Results The mean age was 51 years old and most (78.57%) had locally advanced disease. Five-year OS, PFS, LC, and DMFS in the entire cohort were 57.18% (confidence interval [CI], 34.06%-74.82%), 48.07% (CI, 25.50%-67.52%), 72.11% (CI, 49.96%-85.73%), and 62.85% (CI, 36.33%-80.82%), respectively. There was no significant association between NFkB expression (H-index ≥180) and 3-year and 5-year OS (P-value=0.34), PFS (P-value=0.21), LC (P-value=0.86), or DMFS (P-value=0.18). Conclusions Our study demonstrated that cytoplasmic NFkB-p65 expression (H-index ≥180) was associated with a nonstatistically significant trend toward poor clinical outcomes in locally advanced cervical cancer patients treated definitively with CRT.

BACKGROUND Adenomyosis, characterized by the presence of islands of endometrial tissue surrounded by hypertrophic smooth muscle cells within the myometrium, is one of the most challenging uterine disorders in terms of diagnosis and management. Adenomyosis presents with pelvic pain, excessive uterine bleeding, anemia and infertility. The relative contributions of abnormal endometrial tissue and myometrial smooth muscle cells to the development and growth of adenomyosis are not well understood. Moreover, there is continuing debate on the origins of adenomyosis; two competing theories describe the invagination of basal endometrium into the myometrium or the metaplastic differentiation of remnant endometrial stem/progenitor cells within the myometrium. OBJECTIVE AND RATIONALE A recent series of next-generation sequencing (NGS) studies have provided the best scientific evidence thus far regarding the cellular origins of adenomyosis and the contributions of new signaling pathways to its pathogenesis, survival, and growth. These seminal studies on endometrium, adenomyosis and endometriosis demonstrate or support the following key points. (i) Mutations of KRAS map to both intracavitary endometrial tissue and proximally located adenomyotic samples, supporting the invagination theory of pathogenesis. Driver mutations found in smooth muscle cells of uterine fibroids are absent in adenomyosis. (ii) KRAS and other less frequent mutations are limited to endometrial-type epithelial cells. They are also observed in endometriosis, indicating that the disease process in adenomyosis is similar to that in endometriosis and distinct from that of uterine fibroids. (iii) Activating mutations of KRAS stimulate specific pathways to increase cell survival and proliferation and are associated with progesterone resistance in adenomyosis. Together, these findings suggest that distinct cell populations in eutopic endometrial tissue play key roles in the etiology of adenomyosis. Dependence on ovarian steroids and ovulatory cycles for disease severity is a unique feature of adenomyosis. In this context, common patterns of aberrant gene expression have been reported both in adenomyosis and endometriosis. These include pathways that favor increased estrogen biosynthesis, decreased estradiol metabolism, a unique estrogen receptor beta (ESR2)-driven inflammatory process, and progesterone resistance due to decreased progesterone receptor expression. Since adenomyosis exhibits a uniquely estrogen-driven inflammatory process and progesterone resistance, we discuss the interactions between these molecular characteristics and signaling pathways induced by the newly discovered KRAS mutations. SEARCH METHODS We conducted a comprehensive search using PubMed for human and animal studies published until 2020 in the following areas: adenomyosis, endometriosis, endometrium, NGS, whole-exome sequencing, whole-genome sequencing, RNA sequencing, targeted deep sequencing, epigenetics, driver mutation, KRAS, progesterone resistance, estrogen action and steroid production. OUTCOMES Targeted deep sequencing analyses of epithelial cells in adenomyosis and adjacent basalis endometrial glands demonstrated recurring KRAS mutations in both cell types. This finding suggests that adenomyosis originates from basalis endometrium. Epithelial cells of the endometrium, adjacent adenomyosis and co-occurring endometriosis also share identical KRAS mutations. These findings suggest both adenomyosis and endometriosis are oligoclonal tissues that arise from endometrial cell populations carrying a specific driver mutation that most commonly affects the KRAS gene. WIDER IMPLICATIONS Adenomyosis usually follows an event such as pregnancy that has disrupted the integrity of the endometrial–myometrial junction followed by repetitious menstrual episodes that increase the likelihood of the entrapment of the basalis endometrium within the myometrium. Glandular epithelial cells carrying KRAS mutations and located within the deep crypts of basalis endometrium may become entrapped and invade myometrial tissue to give rise to adenomyosis. Evidence suggests that KRAS mutations may be responsible, in part, for previously observed phenomena such as prolonged cell survival and progesterone resistance in adenomyosis.

Objective To investigate the functional interaction between the Wnt/β-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). Design Laboratory study. Setting Research laboratory. Patient(s) Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. Intervention(s) None. Main Outcome Measure(s) Gene expression, protein phosphorylation, and cell proliferation. Result(s) Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/β-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/β-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated β-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. Conclusion(s) Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent β-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Defining traits of platinum tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum tolerant cells and tumors were identified by using in vitro and in vivo ovarian cancer (OC) models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were found to be enriched in ALDH (+) cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared to parental cells. Additionally, platinum-tolerant cells and tumors highly expressed the Wnt receptor, Frizzled 7 (FZD7). FZD7 knock down improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7(+) from FZD7(-) cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protects cells from chemotherapy-induced oxidative stress. FZD7(+) platinum-tolerant OC cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63 and glutathione metabolism gene sets were strongly correlated in the OC Tumor Cancer Genome Atlas (TCGA) database and in human OC specimens residual after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial stem cell features, characterized by FZD7 expression and dependent on FZD7-β-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype.

Mullerian adenosarcoma (MAS) is a biphasic tumor with malignant stroma. It is most commonly of endometrial origin but occasionally originates in the cervix, ovary, or other pelvic/peritoneal sites. The typical MAS is low grade with an indolent clinical course; however, tumors with sarcomatous overgrowth (SO) or a high-grade sarcoma tend to be aggressive. Tumor etiology is largely unknown. To better understand the global genome alterations and gene mutations in MAS, whole-genome sequencing (WGS) and target validation analysis were performed. MAS showed remarkable chromosome (chr) copy number variation (CNV), specifically, gains in chr 1q, 5p, 12p, 12q, and 17q and losses in chr 3p, 3q, 9p, and 11q. Gain of chr 12q13-15 was present in 50% of cases. The selected gene products in gain regions were upregulated as measured by immunohistochemistry. HMGA2 overexpression was significantly correlated with SO. While the structural variation (SV) rate was relatively low overall, a disproportionally high rate of break-ends at chr 7 was noted involving 6 in-frame rearrangement fusion genes. Among 40 frequently mutated genes detected by WGS and validated in 29 MAS by next generation sequencing (NGS), KMT2C, and BCOR were frequently seen in MAS both with and without SO, while MAGEC1 and KDM6B were strongly associated with SO. Overall, a higher rate of frequently mutated genes was found in MAS with SO (33%) than MAS without (11%). This study uncovers the complex and specific genetic alterations in this malignancy. The findings provide a tool for future investigation of these molecular changes in tumorigenesis and target therapies.

Pseudogenes have long been considered as nonfunctional genomic sequences. Recent studies have shown that they can potentially regulate the expression of protein-coding genes and are dysregulated in diseases including cancer. However, the potential roles of pseudogenes in ovarian cancer have not been well studied. Here we characterized the pseudogene expression profile in HGSOC (high-grade serous ovarian carcinoma) by microarray. We identified 577 dysregulated pseudogenes and most of them were up-regulated (538 of 577). HMGA1P6 (High mobility group AT-hook 1 pseudogene 6) was one of the overexpressed pseudogenes and its expression was inversely correlated with patient survival. Mechanistically, HMGA1P6 promoted ovarian cancer cell malignancy by acting as a ceRNA (competitive endogenous RNA) that led to enhanced HMGA1 and HMGA2 expression. Importantly, HMGA1P6 was transcriptionally activated by oncogene MYC in ovarian cancer. Our findings reveal that MYC may contribute to oncogenesis through transcriptional regulation of pseudogene HMGA1P6 in ovarian cancer.

Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-06.

Uterine leiomyomas (ULM) are histologically and molecularly heterogeneous and clinically they grow at vastly different rates. Several driver gene mutations have been identified in ULM, including MED12 mutations, HMGA2 overexpression, and biallelic FH inactivation. ULM with different driver mutant genes may use different molecular pathways, but currently no clear correlation between gene mutations and growth related pathways has been established. To better define this relationship, we collected ULM with MED12 (n = 25), HMGA2 (n = 15), and FH (n = 27) mutations and examined the sex steroid hormone, cell cycle, and AKT pathway genes by immunohistochemistry. While ER and PR were highly expressed in all types of ULM, FH ULM showed lower ER expression and higher PR expression. HMGA2 tumors had significantly higher levels of AKT signaling and mitogenic activity than other ULM types. HMGA2 activated AKT signaling through upregulation of IGF2BP2. Silencing HMGA2 in ULM cells resulted in downregulation of AKT and upregulation of p16 and p21, which eventually led to cell senescence. HMGA2 overexpression in ULM is not only related to tumor development but also plays a role in controlling cellular proliferation through the AKT pathway.

PARP inhibitors have been widely tested in clinical trials, especially for the treatment of breast cancer and ovarian cancer, and were shown to be highly successful. Because PARP primarily functions in sensing and repairing DNA strand breaks, the therapeutic effect of PARP inhibition is generally believed to be attributed to impaired DNA repair. We here report that oxidative stress is also increased by PARP inhibition and mediates the antitumor effect. We showed that PARP1 is highly expressed in specimens of high grade serous ovarian carcinoma and its activity is required for unperturbed proliferation of ovarian cancer cells. Inhibition or depletion of PARP leads to not only an increase in DNA damage, but also an elevation in the levels of reactive oxygen species (ROS). Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation by PARP inhibition or depletion. We further showed that NADPH oxidases 1 and 4 were significantly upregulated by PARP inhibition and were partially responsible for the induction of oxidative stress. Depletion of NOX1 and NOX4 partially rescued the growth inhibition of PARP1-deficient tumor xenografts. Our findings suggest that in addition to compromising the repair of DNA damage, PARP inhibition or depletion may exert extra antitumor effect by elevating oxidative stress in ovarian cancer cells. Keywords: PARP1, Oxidative stress, NADPH oxidases, Ovarian cancer

Uterine smooth muscle tumors (USMTs) consist of a group of histologically heterogeneous and clinically diverse diseases ranging from malignant leiomyosarcoma (LMS) to benign leiomyoma (ULM). The genetic alterations in LMS are complex, with some genetic alterations present in both LMS and other atypical histologic variants of USMT. In this study, we reviewed 119 USMTs with a diagnosis of LMS, smooth muscle tumor of uncertain malignant potential, atypical leiomyomas/leiomyoma with bizarre nuclei, and cellular leiomyoma, as well as 46 ULMs and 60 myometrial controls. We selected 17 biomarkers highly relevant to LMS in 4 tumorigenic pathways including steroid hormone receptors (estrogen receptor [ER] and progesterone receptor [PR]), cell cycle/tumor suppressor genes, AKT pathway markers, and associated oncogenes. ER and PR expression was significantly lower in LMS than smooth muscle tumor of uncertain malignant potential, atypical leiomyomas/leiomyoma with bizarre nuclei, cellular leiomyoma, and ULM (P .01). Sixty-five percent of LMSs showed complete loss of ER, and 75% of LMSs showed complete loss of PR. All cell cycle genes were differentially expressed in different types of tumor, but significant overlap was noted. More than 75% of LMSs had Ki-67 index greater than 33%, and only 5% in all other types of USMT. Expression of the selected oncogenes varied widely among different types of USMT. PR positivity and p53 had a borderline association with progression-free survival (P = .055 for PR and P = .0847 for p53). Furthermore, high PR expression was significantly associated with a longer overall survival (P = .0163, hazard ratio 0.198). Cell proliferative indices (Ki-67) and sex steroid hormone receptors were the most valuable markers in differentiating LMS from other USMT variants.

The signaling events involved in the onset of ovarian cancer from the fallopian tube epithelium (FTE) are crucial for early detection and treatment of the disease, but they remain poorly defined. Conditional homozygous knockout of PTEN mediated by PAX8-cre recombinase was sufficient to drive endometrioid and serous borderline ovarian carcinoma, providing the first model of FTE-derived borderline tumors. In addition, heterozygous PTEN deletion in the FTE resulted in hyperplasia, providing a model to study early events of human ovarian pathogenesis. To uncover the mechanism underlying the invasion of cancerous oviductal cells to the ovary, PTEN-deficient murine oviductal cells were developed and tagged with green fluorescent protein. Loss of PTEN increased cell migration, invasion, and upregulated WNT4, a key regulator of Müllerian duct development during embryogenesis. Further investigation revealed that WNT4 was required for increased migration and colonization of the ovary by PTEN-deficient oviductal cells in a β-catenin independent manner. Human tumor microarrays and ovarian cancer cells lines confirmed WNT4 expression in cancer and its role in migration. Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis.

// Guannan Zhao 1, 2, * , Qinghui Wang 3, * , Qingqing Gu 1, 2 , Wenan Qiang 4, 5 , Jian-Jun Wei 4 , Peixin Dong 6, 7 , Hidemichi Watari 6, 7 , Wei Li 3 and Junming Yue 1, 2 1 Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, USA 2 Center for Cancer Research, University of Tennessee Health Science Center, Memphis, USA 3 Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, USA 4 Department of Pathology, Department of Obstetrics and Gynecology, Northwestern University School of Medicine, Chicago, USA 5 Center for Developmental Therapeutics, Chemistry of Life Processes Institute, Northwestern University, Evanston, USA 6 Department of Women’s Health Educational System, Hokkaido University School of Medicine, Hokkaido University, Sapporo, Japan 7 Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, Sapporo, Japan * These authors have contributed equally to this work Correspondence to: Junming Yue, email: jyue@uthsc.edu Wei Li, email: wli@uthsc.edu Keywords: BIRC5 (survivin), CRISPR/Cas9 nickase, lentiviral vector, ovarian cancer, epithelial to mesenchymal transition Received: March 29, 2017 Accepted: September 18, 2017 Published: October 17, 2017 ABSTRACT BIRC5 encodes the protein survivin, a member of the inhibitor of apoptosis family. Survivin is highly expressed in a variety of cancers but has very low expression in the corresponding normal tissues, and its expression is often associated with tumor metastasis and chemoresistance. We report that survivin was highly expressed in ovarian cancer and strongly correlated with patient overall poor survival. For the first time, we provide experimental evidence that survivin is involved in epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 gene editing led to the inhibition of EMT by upregulating epithelial cell marker, cytokeratin 7 and downregulating mesenchymal markers: snail2, β-catenin, and vimentin in both ovarian cancer SKOV3 and OVCAR3 cells. Consistent with this molecular approach, pharmacological treatment of ovarian cancer cells using a small molecule survivin inhibitor, YM155 also inhibited EMT in these ovarian cancer cell lines. Overexpression of BIRC5 promoted EMT in SKOV3 cells. Using molecular or pharmacological approaches, we found that cell proliferation, migration, and invasion were significantly inhibited following BIRC5 disruption in both cell lines. Inhibition of BIRC5 expression also sensitized cell responses to paclitaxel treatment. Moreover, loss of BIRC5 expression attenuated TGFβ signaling in both SKOV3 and OVCAR3 cells. Collectively, our studies demonstrated that disruption of BIRC5 expression inhibited EMT by attenuating the TGFβ pathway in ovarian cancer cells.

Activation of mammalian target of rapamycin (mTOR) signaling pathway is associated with poor prognosis of epithelial ovarian cancer. The TSC1-TSC2 complex is a critical negative regulator of mTOR signaling. Here, we demonstrated that TSC1 was frequently downregulated in high-grade serous ovarian carcinoma (HGSOC) and low TSC1 expression level is associated with advanced tumor stage. We next identified miR-130a to be a negative regulator of TSC1 by targeting its 3’UTR. miR-130a was overexpressed in HGSOC and could drive proliferation and invasion/metastasis of ovarian cancer cells. miR-130a could also attenuate rapamycin/starvation-induced autophagy. Ectopic TSC1 expression could block the effects of miR-130a on cell proliferation, migration and autophagy. Finally, we found that miR-130a expression could be upregulated by inflammatory factors and was transactivated by NF-κB. Therefore, our findings establish a crosstalk between inflammation and mTOR signaling that is mediated by miR-130a, which might have a pivotal role in the initiation and progression of HGSOC.

e17513 Background: Nigeria faces a high health burden of cervical cancer (CC), which is worsened by high prevalence of human immunodeficiency virus (HIV) infection. Repetitive elements (RE) are DNA sequences that occur in multiple copies throughout the human genome. HIV infection can lead to RE hypomethylation that causes genome instability, an event often seen in the early phase of tumorigenesis. We aim to examine global RE hypomethylation as a novel epigenetic biomarker for CC among HIV-positive women in Nigeria. Methods: This study involved three groups of women: a) HIV-positive with CC (n=39); b) HIV-positive and cancer-free (n=52); and c) HIV-negative with CC (n=23). We estimated three types of global RE methylation in cervical tissue using genome-wide methylation data: long interspersed nuclear elements (LINE-1), Alu, and human endogenous retrovirus (HERV). We used multiple linear regression adjusting for age, education, parity, employment, cancer stage, body mass index, and sample batch to compare the biomarkers across the HIV/ICC groups and paired t-test to compare 26 pairs of tumor vs. surrounding normal tissues, stratified by HIV status. Receiver operating characteristic curve (ROC) and area under the ROC (AUC) were used to examine the diagnostic value. Results: Among HIV-positive women, all 3 global RE methylation biomarkers were hypomethylated in CC compared to cancer-free (LINE-1: mean difference [MD]=-0.049, p-value=2.9e-8; Alu: MD=-0.011, p-value=2.0e-4; HERV: MD=-0.013, p-value=1.7e-6). Paired analyses showed a larger, more significant MD in HIV-positive stratum than HIV-negative, especially LINE-1 (MD=-0.048 vs. -0.020, p-value=0.004 vs. 0.254). LINE-1 achieved the highest AUC (0.85, 95% CI: 0.76-0.95) in distinguishing tumor tissue from normal tissue among HIV-positive women, followed by HERV (0.82, 95% CI: 0.73-0.92) and Alu (0.60, 95% CI: 0.47-0.72). Conclusions: Global LINE-1 hypomethylation may serve as a novel biomarker for CC screening and early detection for women living with HIV in low- and middle-income countries.

e17504 Background: Invasive cervical cancer (ICC) is one of the HIV-associated cancers with a high burden in Nigeria. ICC occurs at relatively younger age in HIV infected women, with HIV known to promote aging and related diseases, including cancer. DNA methylation changes with increasing age, various health-related exposures, and age-related health outcomes, suggesting a role of DNA methylation in biological aging and disease. We sought to understand the effect of HIV infection on epigenetic age acceleration (EpiAgeAccel) in Nigerian women with ICC. Methods: Epigenetic age (EpiAge) was estimated by Horvath’s calculator using genome-wide methylation data in 116 cervical tissue samples from three groups of women: a) HIV positive with ICC (n=39); b) HIV positive and cancer-free (n=53); and c) HIV negative with ICC (n=24). EpiAgeAccel was computed as the regression residuals of EpiAge against chronological age (ChronAge), representing the independent deviation of EpiAge from ChronAge. We compared EpiAgeAccel across the 3 HIV/ICC groups using multiple linear regressions adjusting for ChronAge, education, parity, employment, cancer stage, body mass index, and study site. Among the ICC women, we compared EpiAgeAccel between 26 tumor tissues and their surrounding normal tissues using paired t-tests, stratified by HIV status. Results: EpiAgeAccel among HIV positive women with ICC was 4.5 years higher than HIV positive and cancer-free women (p=0.019). We did not find substantial differences in EpiAgeAccel between HIV-positive women with ICC and HIV-negative women with ICC. EpiAgeAccel was 7.9 and 2.9 years higher in tumor tissues compared to the surrounding normal tissues among HIV positive women (p=0.021) and negative women (p=0.295), respectively. Conclusions: EpiAge is accelerated in cervical tissue of HIV-infected women with ICC. EpiAgeAccel may be a potential biomarker for ICC screening and early detection for women living with HIV in low- and middle-income countries.

e16137 Background: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality in Nigeria. HIV and viral hepatitis B (HBV) and C (HCV) co-infection are common in Nigeria that significantly accelerates liver disease progression including HCC. Aging-related DNA methylation signatures obtained in liquid biopsy, such as circulating cell-free DNA (ccfDNA) extracted from serum/plasma are promising minimally-invasive biomarkers that may inform HIV-associated HCC. We examined the epigenetic age acceleration (EpiAgeAccel) in ccfDNA in HCC patients with HIV. Methods: The study included three groups of participants: a) HIV positive with HCC (n=7); b) HIV positive and cancer-free (n=45); and c) HIV negative with HCC (n=33). Epigenetic age was estimated by Horvath’s calculator using genome-wide ccfDNA methylation data profiled by Illumina EPIC array. EpiAgeAccel was computed as the residuals of a linear model of epigenetic age on chronological age, namely the unexplained portion of epigenetic age by chronological age. We used multiple linear regression to compare EpiAgeAccel between HIV/HCC groups, adjusting for sex, age, education, alcohol intake, and HBV/HCV infection. Results: Among HIV positive participants, there was a higher percentage of men (57% vs. 22%, p

BACKGROUND Synuclein-γ (SNCG) is highly expressed in advanced solid tumors, including uterine serous carcinoma (USC). The objective of the current study was to determine whether SNCG protein was associated with survival and clinical covariates using the largest existing collection of USCs from the Gynecologic Oncology Group (GOG-8023). METHODS High-density tissue microarrays (TMAs) of tumor tissues from 313 patients with USC were stained by immunohistochemistry for SNCG, p53, p16, FOLR1, pERK, pAKT, ER, PR, and HER2/neu. Associations of SNCG and other tumor markers with overall and progression-free survival were assessed using log-rank tests and Cox proportional-hazards models, which also were adjusted for age, race, and stage. RESULTS The overall survival at 5 years was 46% for women with high SNCG expression and 62% for those with low SNCG expression (log-rank P = .021; hazard ratio [HR], 1.31; 95% confidence interval [CI], 0.91-1.9 in adjusted Cox model). The progression-free survival rate at 5 years was worse for women who had high SNCG expression, at 40%, compared with 56% for those who had low SNCG expression (log-rank P = .0081; HR, 1.36; 95% CI, 0.96-1.92 in adjusted Cox model). High levels of both p53 and p16 were significantly associated with worse overall survival (p53: HR, 4.20 [95% CI, 1.54-11.45]; p16: HR, 1.95 [95% CI, 1.01-3.75]) and progression-free survival (p53: HR, 2.16 [95% CI, 1.09-4.27]; p16: HR, 1.53 [95% CI, 0.87-2.69]) compared with low levels. CONCLUSIONS This largest collection of USCs to date demonstrates that SNCG was associated with poor survival in univariate analyses. SNCG does not predict survival outcome independent of p53 and p16 in models that jointly consider multiple markers. Cancer 2017;123:1144-1155. © 2016 American Cancer Society.

Background Patient derived xenografts (PDX) are generated by transplanting the original patient’s tumor tissue into immune-deficient mice. Unlike xenograft models derived from cell lines, PDX models can better preserve the histopathology from the original patient and molecular pathways. High-grade serous carcinoma (HGSC) is a deadly form of ovarian/fallopian tube cancer whose response to current chemotherapies varies widely due to patient variability. Therefore, a PDX model can provide a valuable tool to study and test treatment options for each individual patient. Methods In this study, over 200 PDX tumors from nine HGSC were analyzed to investigate the nature and behavior of PDX tumors originating from HGSC. PDX tumors were serially passaged (from P0 to P4) and tumors were grafted orthotopically under the ovarian bursa or subcutaneously. Results Comparative analysis of the histology and molecular markers of tumors from over 200 PDX tumor-bearing mice, revealed that the tumors maintained similar histologies, stem cell populations, and expression for the majority of the tested oncogenic markers, compared to the primary tumors. However, a significant loss of steroid hormone receptors and altered expression of immunoresponsive genes in PDX tumors were also noted. Conclusion Our findings provide substantial new information about PDX tumor characteristics from HGSC which will be valuable towards the development of personalized therapy and new drug development for HGSC. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0318-6) contains supplementary material, which is available to authorized users.

While ovarian cancer (OC) is considered as a highly chemoresponsive tumor, 75% women experience tumor relapse associated with chemoresistance. It has been hypothesized that ovarian cancer stem cells (OCSCs) are responsible for developing chemoresistant recurrent tumors and sharing characteristics with normal SCs. Measuring aldehyde dehydrogenase 1 (ALDH1) enzymatic activity has been demonstrated as an established method for OCSC identification and isolation. Our previous studies indicated that while chemotherapy initially “debulks” the mass of OC, residual tumors are enriched in OCSCs. Here, we aimed to identify new markers for CSC enriched platinum-resistant OCs using in vitro and in vivo models. We found that the surviving cells after repeated treatment with platinum were enriched in ALDH(+) cells, formed more spheroids and contained cells expressing CSC-related transcription factors (Sox 2) compared to parental cells, supporting that repeated platinum treatment selects cells with stem-like properties. Next, CSC RT-PCR based platform was used to compare wild-type and chemoresistant OCs and to rule out candidate genes that may drive stemness and chemoresistance. CSC array showed that Frizzled 7 receptor (FZD7), a transmembrane receptor involved in canonical Wnt/β-catenin, emerged as a top highly expressed marker in platinum-resistant compared to parental cells and was next studied as a putative new marker for CSC-enriched chemoresistant OCs. FZD7 expression was increased in platinum-resistant cells compared to parental cells at mRNA and protein levels. FZD7(+) cells were detectable and represent ~2-15% on OC cell lines and cells dissociated from human tumors. FZD7(+) population enriched in the platinum-resistant OCs and xenografts. In addition, KD FZD7 in chemoresistant OC cells restored chemosensitivity of OCs, decreased spheroid formation, and delayed tumor initiation, suggesting the role of FZD7 in regulating chemoresistance and stemness of OCs. RNA sequencing results of FZD7(+)/FZD7(-) OCs revealed that FZD7(+) cells downregulated genes associated to DNA damage response, and upregulated “persister cells” related genes, including EMT and stemness-associated genes. Importantly, mitochondrial dysfunction and oxidative phosphorylation were enriched in FZD7(-) OCs compared with FZD7(+) cells. As “persister cells” have been demonstrated to be dependent on antioxidant protein, glutathione peroxidase 4 (GPX4), for cell survival and maintaining mitochondrial oxidative phosphorylation in other cancer models, we checked GPX4 expression in FZD7(+) OCs and platinum-resistant OCs; the expression of GPX4 is correlated with FZD7 expression associated with chemoresistance. Platinum-resistant cells are more sensitive to GPX4 inhibitor treatment and KD FZD7 decreased GPX4 expression and blunt sensitivity of OCs to GPX4 inhibitor treatment, further supporting that platinum-resistant OCs are “persister like cells,” which are protected by GPX4 from lipid peroxidation stress and ferroptosis. Citation Format: Yinu Wang, Guanyuan Zhao, Hao Huang, Salvatore Condello, Jianjun Wei, Daniela Matei. Role of frizzled-7 in platinum tolerance ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A76.

Approximately one-third of epithelial ovarian tumors exhibit higher levels of c-Met, a receptor tyrosine kinase, activated by Hepatocyte Growth Factor (HGF). c-Met plays an important role in the growth and metastasis of tumor cells. Increased c-Met expression is associated with decreased survival and resistance to chemotherapy in various tumors. More recently, our team identified c-Met overexpression as a mechanism of resistance to neoadjuvant chemotherapy (NACT) in ovarian cancer (OC). The c-Met pathway has been explored as a therapeutic target in various solid tumors, and antibodies targeting c-Met have been proposed as novel anticancer agents. This project aims to investigate the effectiveness of a c-Met monoclonal antibody-drug conjugate (c-Met ADC) in OC models. Here we show that the anti-c-Met antibody alone effectively diminishes c-Met signaling (phosphorylation of c-Met and downstream Akt) but does not inhibit OC cell proliferation. In contrast, the c-Met ADC significantly decreases viability and induces apoptosis in c-Met-expressing OC cells. To validate c-Met expression as a predictor of resistance to NACT in OC, we analyzed c-Met expression in matched pre- and post-treatment samples collected from OC patients undergoing treatment with NACT and in a tissue microarray containing 20 ovarian tumors post-NACT. Detectable c-Met was recorded in ~50% of post-NACT specimens. We evaluated the in vitro antitumor activity of c-Met-ADC on the primary cells isolated from patients, before and after NACT, to assess tumor cell sensitivity to c-Met ADC. The results from these studies support the development of c-Met-ADC as a novel treatment for OC. Citation Format: Anusha Chaparala, Wendy M. Swetzig, Anna E. Strohl, Jian-Jun Wei, Daniela Matei, Shohreh Shahabi. Developing a novel treatment for advanced ovarian cancer by targeting the c-Met pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A36.

Ovarian cancer is the most lethal gynecological malignancy. Patients usually have poor prognosis because of late diagnosis, relapse, and chemoresistance. It is pressing to seek novel agent for the treatment of ovarian cancer. Neferine is a bisbenzylisoquinoline alkaloid isolated from the embryos of lotus (Nelumbo nucifera). In this study, we investigated the antitumor effect of neferine on ovarian cancer cells. We found that neferine exhibited growth-inhibitory effect on human ovarian cancer cells, whereas showing less cytotoxic to non-malignant fallopian tube epithelial cells. Furthermore, we demonstrated that neferine induced autophagy and inactivated the mTOR pathway. Finally, we found that both p38 MAPK and JNK signaling pathways were activated by neferine treatment and contributed to the induction of autophagy in ovarian cancer cells. In conclusion, our findings showed that neferine induced autophagy of human ovarian cancer cells via p38 MAPK/JNK activation. Neferine may be explored as a promising antitumoral agent in ovarian cancer.

High-grade serous cancer (HGSC) is a lethal form of ovarian cancer due to invasion and early metastasis. Gain of epithelial-mesenchymal transition (EMT) contributes to the aggressiveness of HGSC. High-mobility gene group A2 (HMGA2), an architectural transcription factor, plays a major role in HGSC through the regulation of EMT gene expression. Based on the gene profiling analysis, we found that the potent EMT gene, stanniocalcin 2 (STC2), was highly correlated with HMGA2 expression. In the present study, we demonstrated that STC2 was directly regulated by HMGA2 at the transcriptional level. Overexpressing STC2 in vitro directly enhanced cell migration and invasion. To investigate the correlation of STC2 and HMGA2 expression and the potential biomarker for ovarian cancer, three independent large cohorts of ovarian cancer (cohort 1=278, cohort 2=150 and cohort 3=95 cases) were examined in the present study. The results showed that the expression of HMGA2 and STC2 was positively correlated. Furthermore, STC2 expression was significantly associated with tumor grade and histotype. HGSC had significantly higher levels of STC2 expression and was inversely correlated with patient survival. These findings suggested that STC2 is an important new biomarker that can be used for HGSC.

Artesunate, a semi-synthetic derivative of arteminisin originally developed for the treatment of malaria, has recently been shown to possess antitumor properties. One of the cytotoxic effects of artesunate on cancer cells is mediated by induction of oxidative stress and DNA double-strand breaks (DSBs). We report here that in addition to inducing oxidative stress and DSBs, artesunate can also downregulate RAD51 and impair DSB repair in ovarian cancer cells. We observed that the formation of RAD51 foci and homologous recombination repair (HRR) were significantly reduced in artesunate-treated cells. As a consequence, artesunate and cisplatin synergistically induced DSBs and inhibited the clonogenic formation of ovarian cancer cells. Ectopic expression of RAD51 was able to rescue the increased chemosensitivity conferred by artesunate, confirming that the chemosensitizing effect of artesuante is at least partially mediated by the downregulation of RAD51. Our results indicated that artesunatecan compromise the repair of DSBs in ovarian cancer cells, and thus could be employed as a sensitizing agent in chemotherapy.

A deeper understanding of the pathways that drive uterine leiomyoma (ULM) growth and survival requires model systems that more closely mimic the in vivo tumors. This would provide new insights into developing effective therapeutic strategies for these common benign tumors of childbearing-aged women. In this study, we examined the role of BCL-2 in mediating ULM survival in the context of increased protein kinase B (AKT) and oxidative stress using a three-dimensional (3D), spheroid-based model that more closely resembles the native ULM tumor microenvironment. Human primary cells from matched myometrium (MM) and ULM tissues were used to establish spheroid cultures in vitro. Histological and immunohistochemical methods were used to assess the spheroid architecture and characteristics. Viability assays for 3D cultures were used to evaluate their response to BH3 mimetics and the superoxide inducer, paraquat (PQ). Primary MM and ULM cells formed spheroids in culture. Notably, ULM spheroids exhibited low proliferation, increased oxidative stress, and secretion of interstitial collagen. Knockdown studies revealed that AKT sustained BCL-2 expression in ULM. The targeting of BCL-2 with BH3 mimetics effectively reduced viability and induced apoptosis in a subset of ULM spheroids. ULM spheroids that did not respond to BH3 mimetics alone responded to combination treatment with PQ. In conclusion, BCL-2 mediates AKT survival of ULM, providing compelling evidence for further evaluation of BH3 mimetics for ULM treatment. ULM spheroids recapitulated intrinsic features of the native ULM tumor microenvironment and can be used as a model for preclinical testing of potential therapeutic options for ULM.

// Andrea E. Murmann 1 , Kaylin M. McMahon 3,6 , Ashley Haluck-Kangas 1 , Nandini Ravindran 1 , Monal Patel 1 , Calvin Y. Law 1 , Sonia Brockway 1 , Jian-Jun Wei 4 , C. Shad Thaxton 3,5,6,7 and Marcus E. Peter 1,2,5 1 Department of Medicine/Division of Hematology/Oncology, Feinberg School of Medicine, Chicago, IL, USA 2 Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Chicago, IL, USA 3 Department of Urology, Feinberg School of Medicine, Chicago, IL, USA 4 Department of Pathology and Obstetrics and Gynecology, Feinberg School of Medicine, Chicago, IL, USA 5 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University, Chicago, IL, USA 6 Simpson Querrey Institute (SQI) for BioNanotechnology, Chicago, IL, USA 7 International Institute for Nanotechnology, Evanston, IL, USA Correspondence to: Marcus E. Peter, email: // Keywords : RNAi, Fas, ovarian cancer, TLP nanoparticles, cell death Received : January 23, 2017 Accepted : September 13, 2017 Published : October 04, 2017 Abstract The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3’UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro , TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo , two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

High-grade serous ovarian carcinoma (HGSOC), the most common and aggressive subtype of epithelial ovarian cancer, is characterized by TP53 mutations and genetic instability. Using miRNA profiling analysis, we found that miR-145, a p53 regulated miRNA, was frequently down-regulated in HGSOC. miR-145 down-regulation was further validated in a large cohort of HGSOCs by qPCR. Overexpression of miR-145 in ovarian cancer cells significantly suppressed proliferation, migration and invasion in vitro and inhibited tumor growth and metastasis in vivo. Metadherin (MTDH) was subsequently identified as a direct target of miR-145, and was found to be significantly up-regulated in HGSOC. Furthermore, overexpression of MTDH rescued the inhibitory effects of miR-145 in ovarian cancer cells. Finally, we found that high level of MTDH expression correlated with poor prognosis of HGSOC. Therefore, lack of suppression of MTDH by miR-145 when p53 is dysfunctional leads to increased tumor growth and metastasis of HGSOC. Our study established a new link between p53, miR-145 and MTDH in the regulation of tumor growth and metastasis in HGSOC.

Recent identification of somatic MED12 mutations in most uterine leiomyomas brings a new venue for the study of the tumorigenesis of leiomyomas. We are particularly interested in the correlation of MED12 and HMGA2 gene products in leiomyomas and leiomyosarcomas with and without MED12 mutations. To address these issues, in this study we examined MED12 mutations in a large cohort of usual type leiomyomas (178 cases) and uterine leiomyosarcomas (32 cases). We found that 74.7% (133/178) of leiomyomas had MED12 mutations, which was consistent with several independent studies. In contrast, only 9.7% (3/32) of leiomyosarcomas harbored MED12 mutations. Expression analysis by western blot and immunohistochemistry revealed that those leiomyomas with complex MED12 mutations had significantly lower protein products than the matched myometrium. Interestingly, most leiomyosarcomas without MED12 mutations also had very low levels of MED12 expression in comparison to the matched myometrium. These findings suggest a potential functional role of MED12 in both benign and malignant uterine smooth muscle tumors. When we further examined HMGA2 expression in all leiomyomas and leiomyosarcomas, we found that HMGA2 overexpression was exclusively present in those leiomyomas with no MED12 mutation, accounting for 10.1% (18/178) of total leiomyomas and 40% (18/45) of non-MED12 mutant leiomyomas. Twenty-five percent (8/32) of leiomyosarcomas had HMGA2 overexpression, and no MED12 mutations were found in HMGA2 positive leiomyosarcoma. These findings strongly suggest that MED12 mutations and HMGA2 overexpression are independent genetic events that occur in leiomyomas, and they may act differently in the tumorigenesis of uterine leiomyomas.

High-grade serous ovarian carcinoma (HGSOC) is a fatal disease, and its grave outcome is largely because of widespread metastasis at the time of diagnosis. Current chemotherapies reduce tumor burden, but they do not provide long-term benefits for patients with cancer. The aggressive tumor growth and metastatic behavior characteristic of these tumors demand novel treatment options such as anti-microRNA treatment, which is emerging as a potential modality for cancer therapy. MicroRNA-182 (miR182) overexpression contributes to aggressive ovarian cancer, largely by its negative regulation of multiple tumor suppressor genes involved in tumor growth, invasion, metastasis, and DNA instability. In this study, we examined the therapeutic potential of anti-miR182 utilizing the animal orthotopic model to mimic human ovarian cancer using ovarian cancer cells SKOV3 (intrabursal xenografts) and OVCAR3 (intraperitoneal injection). These models provide a valuable model system for the investigation of ovarian cancer therapy in vivo. Through a combination of imaging, histological, and molecular analyses, we found that anti-miR182 treatment can significantly reduce tumor burden (size), local invasion, and distant metastasis compared with its control in both models. The bases of anti-miR182 treatment are mainly through the restoration of miR182 target expression, including but not limited to BRCA1, FOXO3a, HMGA2, and MTSS1. Overall, our results strongly suggest that anti-miR182 can potentially be used as a therapeutic modality in treating HGSOC. Mol Cancer Ther; 13(7); 1729–39. ©2014 AACR.

BACKGROUND: Uterine smooth muscle tumors (USMTs) constitute a group of histologic, genetic, and clinical heterogeneous tumors that include at least 6 major histologically defined tumor types: leiomyoma (ULM), mitotically active leiomyoma (MALM), cellular leiomyoma (CLM), atypical leiomyoma (ALM), uncertain malignant potential (STUMP), and leiomyosarcoma (LMS). Apart from ULM and LMS, the nature of these variants is not well defined. METHODS: A total of 167 cases of different USMT variants were collected, reviewed, and diagnostically confirmed based on the World Health Organization and Stanford schemes. These included 38 cases of LMS, 18 cases of STUMP, 42 cases of ALM, 22 cases of CLM, 7 cases of MALM, and 40 cases of ULM. Molecular analysis included selected microRNAs (miRNAs), oncogenes, and tumor suppressors that are highly relevant to USMT. RESULTS: Overall, 49% (17/35) of LMS cases and 7% (1/14) of STUMP cases died due to their USMT, but no deaths were attributed to ALM. miRNA profiling revealed that ALM and LMS shared similar miRNA signatures. P53 mutations and PTEN deletions were significantly higher in LMS, ALM, and STUMP compared with other USMT variants (P 74%) but were significantly less common (

SIGNIFICANCE: High Grade Serous Cancer (HGSOC) Requires Novel Biomarkers for early detection. Survival rates for HGSOC patients have not substantially improved, over the past decades, mainly because the disease is only detected at the metastatic stage. The findings of early pre-malignant lesions in the fallopian tube, but not in the ovary, suggested that HGSOCs may originate from the fallopian tube epithelium (FTE) and metastasize to the ovary. Studying the mechanisms of HGSOC genesis from the correct cell of origin is critical to the early detection of the disease. Our main goal is to determine how genetic and molecular alterations contribute to the early events of HGSOC genesis from FTE. Recent transgenic animal models of FTE-derived HGSOC have pointed out the critical role of loss of PTEN (phosphatase and tensin homolog) from FTE in generating HGSOC when in combination with p53 mutation and alteration of BRCA1/2, but not by itself. Our lab was the first to show that deletion of PTEN alone in FTE is sufficient to lead to tumor formation and peritoneal dissemination suggesting that the importance of PTEN in the early events of the tumorigenic cascade may have been underestimated. Loss of PTEN from FTE is sufficient to drive ovarian cancer, however, the key signaling driving tumorigenesis when PTEN expression is low remain poorly defined and yet pivotal to new treatment options given its common occurrence. Studying the progression of HGSOC genesis from FTE is essential to refining the strategy for targeted therapies and to discovering novel biomarkers for early detection of the disease. PURPOSE: to identify signaling pathways and drug response of different subpopulations of tumorigenic cells generated upon PTEN deletion. RESULTS: Loss of PTEN in the FTE upregulates markers of cancer stem cells (CSC) such as WNT4, LGR5, ALDH1, c-Kit, CD44, and Dll4. We also discovered that the CSC markers are confined in a specific subpopulation of cells with increased diameter that we call CSCHigh as compared to a smaller population that we call CSCLow. In fact, when these two populations are separated by flow cytometry based sorting, the CSCHigh retains all the CSC markers. In addition, using a limiting dilution assay, we found that the CSCHigh subpopulation forms tumor at a faster rate in vivo. In addition, CSCHigh forms bigger spheroids and are more resistant to chemotherapeutic agents, suggesting that more targeted therapy may be developed for these stem-like, tumorigenic subpopulations. We also show that loss of PAX2 is mediating the CSCHigh phenotype suggesting that therapies to re-express PAX2 may prevent development of HGSOC. CONCLUSIONS: Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis. Citation Format: Angela Russo, Austin A. Czarnecki, Matthew Dean, Dan Lantvit, Jian-Jun Wei Joanna E. Burdette. STUDYING THE SIGNALING PATHWAYS OF DISTINCT SUBPOPULATIONS OF CELLS GENERATED UPON PTEN DELETION FROM FTE AND IDENTIFICATION OF MARKERS OF EARLY TUMORIGENESIS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-012.

Soft tissue sarcomas are a heterogeneous group of tumors associated with poor clinical outcome. Although a subset of soft tissue sarcomas is characterized by simple karyotypes and recurrent chromosomal translocations, the mechanisms driving cytogenetically complex sarcomas are largely unknown. Clinical evidence led us to partially inactivate Pten and Tp53 in the smooth muscle lineage of mice, which developed high-grade undifferentiated pleomorphic sarcomas, leiomyosarcomas, and carcinosarcomas that widely recapitulate the human disease, including the aberrant karyotype and metastatic behavior. Pten was found haploinsufficient, whereas the wild-type allele of Tp53 invariably gained point mutations. Gene expression profiles showed up-regulated Notch signaling in Pten(Δ/+)Tp53(Δ/+) tumors compared with Pten(+/+)Tp53(Δ/+) tumors. Consistently, Pten silencing exacerbated the clonogenic and invasive potential of Tp53-deficient bone marrow-derived mouse mesenchymal stem cells and tumor cells and activated the Notch pathway. Moreover, the increased oncogenic behavior of Pten(Δ/+)Tp53(Δ/+) and shPten-transduced Pten(+/+)Tp53(Δ/+) tumor cells was counteracted by treatment with a γ-secretase inhibitor, suggesting that the aggressiveness of those tumors can be attributed, at least in part, to enhanced Notch signaling. This study demonstrates a cooperative role for Pten and Tp53 suppression in complex karyotype sarcomas while establishing Notch as an important functional player in the cross talk of these pathways during tumor progression. Our results highlight the importance of molecularly subclassifying patients with high-grade sarcoma for targeted treatments.

Context Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective To define differential gene expression and signaling pathways in leiomyoma cell populations. Design Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting Research laboratory. Patients Eight African American women. Interventions None. Main Outcomes Measures Gene expression patterns, cell proliferation, and differentiation. Results A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Acetylation-mediated inactivation of MnSOD triggers activation of prosurvival AKT signaling in uterine smooth muscle tumors., AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM’s prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬

Smooth muscle (SM) is a spontaneously contractile tissue that provides physical support and function to organs such as the uterus. Uterine smooth muscle–related neoplasia comprise common well-differentiated benign lesions called leiomyomas (ULM), and rare, highly aggressive and pleomorphic tumors named leiomyosarcomas (ULMS). MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in normal cellular development and tissue homeostasis that can be used to accurately subclassify different tumor types. Here, we demonstrate that miRNAs are required for full smooth muscle cell (SMC) differentiation of bone marrow–derived human mesenchymal stem cells (hMSCs). We also report a miRNA signature associated with this process. Moreover, we show that this signature, along with miRNA profiles for ULMS and ULM, are able to subclassify tumors of smooth muscle origin along SM differentiation. Using multiple computational analyses, we determined that ULMS are more similar to hMSCs as opposed to ULM, which are linked with more mature SMCs and myometrium. Furthermore, a comparison of the SM differentiation and ULMS miRNA signatures identified miRNAs strictly associated with SM maturation or transformation, as well as those modulated in both processes indicating a possible dual role. These results support separate origins and/or divergent transformation pathways for ULM and ULMS, resulting in drastically different states of differentiation. In summary, this work expands on our knowledge of the regulation of SM differentiation and sarcoma pathogenesis.

Inflammatory breast cancer (IBC) is the most aggressive clinical manifestation of breast cancer (BC), with striking erythema of the overlying skin, and exhibiting features of early metastatic and treatment-refractory disease. Malignant cells and infiltrating leukocytes, e.g. tumor-associated microphages (TAMs) commonly designated "M2" macrophages, play a major part in metastasis and treatment resistance. TAMs help promote tumor growth and progression. There is a significant need for preclinical models that retain the characteristics of the original patient tumor for investigating mechanisms of IBC metastasis and for testing candidate therapies. Six samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from five IBC or metastatic breast cancer (MBC) patients were obtained from Northwestern Memorial Hospital. IBC cells were used for 3D tumor spheroid cultures, and for developing PDX tumor models in immunodeficient mice. Short Tandem Repeat profiling against with original patient tumor DNA was conducted to establish patient DNA identify, and to serve as a reference for authenticating derivative tumor models. The tumor genomic mutation analysis indicated the most common alterations of five IBC or MBC patients were TP53 (75%), CCND1 (75%), BRCA2 (50%), NF1 (50%), FGFR1 (50%), CDK6 (50%), EGFR (50%), MYC (50%), BRAF (50%). Examination IBC pleural effusion cellular composition indicated ~60% were tumor cells and ~40% were M2 TAMs (CD163+). In early 3D spheroid IBC culture, a symbiotic phenomenon was observed between tumor cells and attached TAMs; Anti-cancer drugs GW2580 and Pexidartinib inhibited 32% and 68% of cell proliferation, respectively, when added to spheroid cultures at 100 nanomolar. Histopathologic analysis of derivative IBC and MBC PDX revealed highly heterogeneous characteristics and metastatic features characteristic of corresponding patient tumors. Subcutaneous tumors also demonstrated metastasis to liver and lung tissue. NSG mice engrafted with original IBC pleural effusion cells or derived spheroid cultures manifested a variety of inflammatory clinical symptoms. Mice engrafted with original pleural effusion cells developed palpable tumor and manifested most IBC-characteristic including reddish and ulcerated skin lesions necessitating euthanasia within 2 months of cell engraftment. Mice engrafted with > 6 passage or Our results indicate IBC 3D spheroid cultures and PDX tumor models recapitulate clinical characteristics observed in IBC patients, and will prove useful for studying mechanisms of IBC metastasis as well as responsiveness to investigational therapies. Citation Format: Wenan Qiang, Youbin Zhang, Demirkan Gursel Gursel, Jian-Jun Wei, Charles David James, Thomas V. O'Halloran, Massimo Cristofanilli. Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1046.

Ovarian carcinoma consists of a group of histologically heterogeneous diseases involving distinct tumorigenic pathways. High-grade papillary serous carcinoma of the ovary is commonly associated with p53 mutations. HMGA2, an oncofetal protein, is found to be overexpressed in ovarian cancer. To study the function of HMGA2 in ovarian cancer, it is important to know which subtypes of ovarian cancer are associated with HMGA2 overexpression. In this study, we collected six different types of ovarian cancer and examined HMGA2 expression by immunohistochemistry, along with HMGA1, p53 and Ki-67. We found that HMGA2 overexpression was significantly higher in high-grade papillary serous carcinoma (64%) and carcinosarcoma (60%) than in other types of ovarian cancers (7-23%). HMGA2 overexpression was moderately associated with dominant p53 mutations (R=0.51). In addition, the microRNA in situ analysis revealed that let-7b, the HMGA2-negative regulators, were significantly lost in high-grade serous carcinoma. Our findings suggest that HMGA2 is an important molecular change significantly related to high-grade papillary serous carcinoma and is less common in other histological types of ovarian cancer.

Before high-grade papillary serous carcinoma (HG-PSC) becomes invasive, it is believed to be a poorly defined short-lived precursor lesion. A recent characterization of serous tubal intraepithelial carcinoma (STIC) and of the p53 signature suggested that HG-PSC may follow a stepwise progression on cellular and molecular levels. High-mobility group AT-hook 2 (HMGA2), an oncofetal protein, is overexpressed in ovarian cancer. To test whether HMGA2 can be another valuable marker for STIC, we examined HMGA2 expression in 3 groups of patients: (1) 24 patients with STIC and its invasive counterpart, HG-PSC of the fallopian tubes, (2) 24 patients with HG-PSC of the ovaries but without STIC (positive control), and (3) 30 patients with cancer and normal fallopian tubes (negative control). We found that HMGA2 was overexpressed in 75% of patients with STIC, was coexpressed with p53 in more than 50% of patients, and was completely negative in the secretory cells of the 30 patients with normal fallopian tubes. Among 7 patients with cells negative for p53 staining, HMGA2 was positive in 5; among 6 patients whose tumor cells were negative for HMGA2 in STIC, 3 were positive for HMGA2 in the invasive component; about 70% of invasive HG-PSC tumor cells were immunoreactive for both HMGA2 and TP53. In invasive carcinoma, HMGA2 overexpression was correlated with p53 (r=0.45), indicating the role of HMGA2 in p53 mediated tumor progression. Our findings of immunoreactivity for HMGA2 may lead to a novel, useful biomarker to complement p53 in the detection of early-stage serous carcinoma.

A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease. [Cancer Res 2009;69(8):3332–8]

Overexpression of HMGA2 is common in uterine leiomyomas (ULM). The expression of HMGA2 in its malignant counterpart – uterine leiomyosarcomas (ULMS) remains undetermined. Recently it has been shown that repression of HMGA2 by microRNA let-7s is a critical molecular regulatory mechanism associated with tumour growth in many tumours and cell types, including leiomyomas. To test whether HMGA2 and let-7s play a role in ULMS, we examined the levels of endogenous HMGA2 and let-7 expression and found a significant correlation between these two molecules in a case-matched cohort of human ULMS. We found that overexpression of HMGA2 and let-7-mediated HMGA2 repression is a relevant molecular alteration in ULMS. Disrupting the control of HMGA2 and let-7 pairs promotes ULMS cell growth in vitro.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOE(LOW)) was developed and continuously passaged in culture to mimic cellular aging (MOE(HIGH)). The MOE(HIGH) cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOE(HIGH) cells proliferated significantly faster than MOE(LOW), and the MOE(HIGH) cells produced more 2D foci and 3D soft agar colonies as compared to MOE(LOW) MOE(HIGH) were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOE(HIGH) and MOE(LOW) were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOE(HIGH) had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOE(HIGH) were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.