PROTACs: Current and Future Potential as a Precision Medicine Strategy to Combat Cancer.

Proteolysis targeting chimeras (PROTAC) are an emerging precision medicine strategy, which targets key proteins for proteolytic degradation to ultimately induce cancer cell killing. These hetero-bifunctional molecules hijack the ubiquitin proteasome system to selectively add polyubiquitin chains onto a specific protein target to induce proteolytic degradation. Importantly, PROTACs have the capacity to target virtually any intracellular and transmembrane protein for degradation, including oncoproteins previously considered undruggable, which strategically positions PROTACs at the crossroads of multiple cancer research areas. In this review, we present normal functions of the ubiquitin regulation proteins and describe the application of PROTACs to improve the efficacy of current broad-spectrum therapeutics. We subsequently present the potential for PROTACs to exploit specific cancer vulnerabilities through synthetic genetic approaches, which may expedite the development, translation, and utility of novel synthetic genetic therapies in cancer. Finally, we describe the challenges associated with PROTACs and the ongoing efforts to overcome these issues to streamline clinical translation. Ultimately, these efforts may lead to their routine clinical use, which is expected to revolutionize cancer treatment strategies, delay familial cancer onset, and ultimately improve the lives and outcomes of those living with cancer.

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas.

OBJECTIVE: Inhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. METHODS: A recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. RESULTS: Loss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. CONCLUSIONS: NF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

Navigating the Blood-Brain Barrier: Challenges and Therapeutic Strategies in Breast Cancer Brain Metastases.

Breast cancer (BC) is the most common cancer in women, with metastatic BC being responsible for the highest number of deaths. A frequent site for BC metastasis is the brain. Brain metastasis derived from BC involves the cooperation of multiple genetic, epigenetic, angiogenic, and tumor-stroma interactions. Most of these interactions provide a unique opportunity for development of new therapeutic targets. Potentially targetable signaling pathways are Notch, Wnt, and the epidermal growth factor receptors signaling pathways, all of which are linked to driving BC brain metastasis (BCBM). However, a major challenge in treating brain metastasis remains the blood-brain barrier (BBB). This barrier restricts the access of unwanted molecules, cells, and targeted therapies to the brain parenchyma. Moreover, current therapies to treat brain metastases, such as stereotactic radiosurgery and whole-brain radiotherapy, have limited efficacy. Promising new drugs like phosphatase and kinase modulators, as well as BBB disruptors and immunotherapeutic strategies, have shown the potential to ease the disease in preclinical studies, but remain limited by multiple resistance mechanisms. This review summarizes some of the current understanding of the mechanisms involved in BC brain metastasis and highlights current challenges as well as opportunities in strategic designs of potentially successful future therapies.

Development and validation of a prognostic 15-gene signature for stratifying HER2+/ER+ breast cancer.

Background: Human epidermal growth receptor 2-positive (HER2+) breast cancer (BC) is a heterogeneous subgroup. Estrogen receptor (ER) status is emerging as a predictive marker within HER2+ BCs, with the HER2+/ER+ cases usually having better survival in the first 5 years after diagnosis but have higher recurrence risk after 5 years compared to HER2+/ER-. This is possibly because sustained ER signaling in HER2+ BCs helps escape the HER2 blockade. Currently HER2+/ER+ BC is understudied and lacks biomarkers. Thus, a better understanding of the underlying molecular diversity is important to find new therapy targets for HER2+/ER+ BCs. Methods: In this study, we performed unsupervised consensus clustering together with genome-wide Cox regression analyses on the gene expression data of 123 HER2+/ER+ BC from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) cohort to identify distinct HER2+/ER+ subgroups. A supervised eXtreme Gradient Boosting (XGBoost) classifier was then built in TCGA using the identified subgroups and validated in another two independent datasets (Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and Gene Expression Omnibus (GEO) (accession number GSE149283)). Computational characterization analyses were also performed on the predicted subgroups in different HER2+/ER+ BC cohorts. Results: We identified two distinct HER2+/ER+ subgroups with different survival outcomes using the expression profiles of 549 survival-associated genes from the Cox regression analyses. Genome-wide gene expression differential analyses found 197 differentially expressed genes between the two identified subgroups, with 15 genes overlapping the 549 survival-associated genes.XGBoost classifier, using the expression values of the 15 genes, achieved a strong cross-validated performance (Area under the curve (AUC) = 0.85, Sensitivity = 0.76, specificity = 0.77) in predicting the subgroup labels. Further investigation partially confirmed the differences in survival, drug response, tumor-infiltrating lymphocytes, published gene signatures, and CRISPR-Cas9 knockout screened gene dependency scores between the two identified subgroups. Conclusion: This is the first study to stratify HER2+/ER+ tumors. Overall, the initial results from different cohorts showed there exist two distinct subgroups in HER2+/ER+ tumors, which can be distinguished by a 15-gene signature. Our findings could potentially guide the development of future precision therapies targeted on HER2+/ER+ BC.

Aberrant HMGA2 Expression Sustains Genome Instability That Promotes Metastasis and Therapeutic Resistance in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide, accounting for nearly ~10% of all cancer diagnoses and deaths. Current therapeutic approaches have considerably increased survival for patients diagnosed at early stages; however, ~20% of CRC patients are diagnosed with late-stage, metastatic CRC, where 5-year survival rates drop to 6-13% and treatment options are limited. Genome instability is an enabling hallmark of cancer that confers increased acquisition of genetic alterations, mutations, copy number variations and chromosomal rearrangements. In that regard, research has shown a clear association between genome instability and CRC, as the accumulation of aberrations in cancer-related genes provides subpopulations of cells with several advantages, such as increased proliferation rates, metastatic potential and therapeutic resistance. Although numerous genes have been associated with CRC, few have been validated as predictive biomarkers of metastasis or therapeutic resistance. A growing body of evidence suggests a member of the High-Mobility Group A (HMGA) gene family, HMGA2, is a potential biomarker of metastatic spread and therapeutic resistance. HMGA2 is expressed in embryonic tissues and is frequently upregulated in aggressively growing cancers, including CRC. As an architectural, non-histone chromatin binding factor, it initiates chromatin decompaction to facilitate transcriptional regulation. HMGA2 maintains the capacity for stem cell renewal in embryonic and cancer tissues and is a known promoter of epithelial-to-mesenchymal transition in tumor cells. This review will focus on the known molecular mechanisms by which HMGA2 exerts genome protective functions that contribute to cancer cell survival and chemoresistance in CRC.

Exploring the role of a multidisciplinary hereditary gynecologic oncology clinic in epithelial ovarian cancer risk-reducing surgical decision-making practices: A mixed-methods study.

Individuals that have gynecologic reproductive organs with pathogenic variants in BRCA1 or BRCA2 ("BRCA-positive") have an increased risk of developing high-grade serous ovarian cancer (HGSOC). The majority of HGSOC develops in the fallopian tubes and later spreads to the ovaries and peritoneal cavity. Therefore, risk-reducing salpingo-oophorectomy (RRSO) is recommended for those who are BRCA-positive to preventatively remove their ovaries and fallopian tubes. The Hereditary Gynecology Clinic (HGC) is a provincial program in Winnipeg, Canada, that specifically targets care to the unique needs of such individuals through an interdisciplinary team of gynecological oncologists, menopause specialists, and registered nurses. A mixed-methods study design was used to explore the decision-making processes of these BRCA-positive individuals who have been recommended (or who completed) RRSO and experiences with healthcare providers at the HGC influenced this decision. Individuals who are BRCA-positive without a previous diagnosis of HGSOC and who had previously received genetic counselling were recruited from the HGC and the provincial cancer genetics program (Shared Health Program of Genetics & Metabolism). Forty-three people completed a survey and 15 participated in an in-depth interview about their experiences and decisions surrounding RRSO. Surveys were analyzed to compare scores on validated scales related to decision-making and cancer-related worry. Qualitative interviews were transcribed, coded, and analyzed using interpretive description. Participants described the complex decisions faced by those who are BRCA-positive, which are intertwined with life experiences and circumstances including age, marital status, and family disease history. Participants interpreted their HGSOC risk through a personalized "lens" of contextual factors that impacted perceptions about the practical and emotional implications of RRSO and the need for surgery. Mean scores on validated scales evaluating the HGC's impact on decisional outcomes and preparedness for decision-making about RRSO were not significant, indicating that the HGC played a supportive role, rather than helping with decision-making itself. Therefore, we present a novel framework that consolidates the various influences on decision-making and connects them to the psychological and practical implications of RRSO in the context of the HGC. Strategies for improving support, decisional outcomes, and the overall experiences of individuals who are BRCA-positive attending the HGC are also described.

Referral, Genetic Counselling, and BRCA Testing in the Manitoba High-Grade Serous Ovarian Cancer Population, 2004-2019.

(1) Background: The primary objective of this study was to examine the rate of genetic referral, BRCA testing, and BRCA positivity amongst all patients with high-grade serous ovarian cancers (HGSOC) from 2004-2019. The secondary objective was to analyze secondary factors that may affect the rates of referral and testing. (2) Methods: This population-based cohort study included all women diagnosed with HGSOC using the Manitoba Cancer Registry, CervixCheck registry, Medical Claims database at Manitoba Health, the Hospital Discharge abstract, the Population Registry, and Winnipeg Regional Health Authority genetics data. Data were examined for three different time cohorts (2004-2013, 2014-2016; 2017-2019) correlating to practice pattern changes. (3) Results: A total of 944 patients were diagnosed with HGSOC. The rate of genetic referrals changed over the three timeframes (20.0% → 56.7% → 36.6%) and rate of genetic testing increased over the entire timeframe. Factors found to increase rates of referral and testing included age, histology, history of oral contraceptive use, and family history of ovarian cancer. Prior health care utilization indicators did not affect genetic referral or testing. (4) Conclusion: The rate of genetic referral (2004-2016) and BRCA1/2 testing (2004-2019) for patients with a diagnosis of HGSOC increased over time. A minority of patients received a consultation for genetics counselling, and even fewer received testing for a BRCA1/2. Without a genetic result, it is difficult for clinicians to inform treatment decisions. Additional efforts are needed to increase genetics consultation and testing for Manitoban patients with HGSOC. Effects of routine tumour testing on rates of genetic referral will have to be examined in future studies.

Reduced SKP2 Expression Adversely Impacts Genome Stability and Promotes Cellular Transformation in Colonic Epithelial Cells.

Despite the high morbidity and mortality rates associated with colorectal cancer (CRC), the underlying molecular mechanisms driving CRC development remain largely uncharacterized. Chromosome instability (CIN), or ongoing changes in chromosome complements, occurs in ~85% of CRCs and is a proposed driver of cancer development, as the genomic changes imparted by CIN enable the acquisition of karyotypes that are favorable for cellular transformation and the classic hallmarks of cancer. Despite these associations, the aberrant genes and proteins driving CIN remain elusive. SKP2 encodes an F-box protein, a variable subunit of the SKP1-CUL1-F-box (SCF) complex that selectively targets proteins for polyubiquitylation and degradation. Recent data have identified the core SCF complex components (SKP1, CUL1, and RBX1) as CIN genes; however, the impact reduced SKP2 expression has on CIN, cellular transformation, and oncogenesis remains unknown. Using both short- small interfering RNA (siRNA) and long-term (CRISPR/Cas9) approaches, we demonstrate that diminished SKP2 expression induces CIN in both malignant and non-malignant colonic epithelial cell contexts. Moreover, temporal assays reveal that reduced SKP2 expression promotes cellular transformation, as demonstrated by enhanced anchorage-independent growth. Collectively, these data identify SKP2 as a novel CIN gene in clinically relevant models and highlight its potential pathogenic role in CRC development.

Aberrant SKP1 Expression: Diverse Mechanisms Impacting Genome and Chromosome Stability.

The S-phase Kinase-Associated Protein 1 (SKP1) is a core component of the SKP1, Cullin 1, F-box protein (SCF) complex, an E3 ubiquitin ligase that serves to poly-ubiquitinate a vast array of protein targets as a signal for their proteasomal degradation, thereby playing a critical role in the regulation of downstream biological processes. Many of the proteins regulated by SKP1 and the SCF complex normally function within pathways that are essential for maintaining genome stability, including DNA damage repair, apoptotic signaling, and centrosome dynamics. Accordingly, aberrant SKP1 and SCF complex expression and function is expected to disrupt these essential pathways, which may have pathological implications in diseases like cancer. In this review, we summarize the central role SKP1 plays in regulating essential cellular processes; we describe functional models in which SKP1 expression is altered and the corresponding impacts on genome stability; and we discuss the prevalence of SKP1 somatic copy number alterations, mutations, and altered protein expression across different cancer types, to identify a potential link between SKP1 and SCF complex dysfunction to chromosome/genome instability and cancer pathogenesis. Ultimately, understanding the role of SKP1 in driving chromosome instability will expand upon our rudimentary understanding of the key events required for genome/chromosome stability that may aid in our understanding of cancer pathogenesis, which will be critical for future studies to establish whether SKP1 may be useful as prognostic indicator or as a therapeutic target.

DREAM, a possible answer to the estrogen paradox of the Women's Health Initiative Trial.

Estrogen is thought to cause proliferation of all estrogen receptor positive (ER+) breast cancers. Paradoxically, in the Women's Health Initiative Trial, estrogen-only hormone replacement therapy reduced the incidence and mortality of low grade, ER+, HER2- breast cancer. We gave estradiol to 19 post-menopausal women with newly diagnosed low-grade, ER+, HER2- breast cancer in a prospective window of opportunity clinical trial and examined the changes in proliferation and gene expression before and after estradiol treatment. Ki67 decreased in 13/19 (68%) patients and 8/13 (62%) showed a decrease in Risk of Recurrence Score. We chose three prototypical estrogen responders (greatest decrease in ROR) and non-responders (no/minimal change in ROR) and applied a differential gene expression analysis to develop pre-treatment (PRESTO-30core) and post-treatment (PRESTO-45surg) gene expression profiles. The PRESTO-30core predicted adjuvant benefit in a published series of tamoxifen, the partial estrogen agonist. Of the 45 genes in the PRESTO-45surg, thirty contain the Cell cycle genes Homology Region (CHR) motif that binds the class B multi-vulva complex (MuvB) a member of the DREAM (Dimerization partner, retinoblastoma-like proteins, E2F, MuvB) complex responsible for reversible cell cycle arrest or quiescence. There was also near uniform suppression (89%) of the remaining DREAM genes consistent with estrogen induced activation of the DREAM complex to mediate cell cycle block after a short course of estrogens. To our knowledge, this is the first report to show estrogen modulation of DREAM genes and suggest involvement of DREAM pathway associated quiescence in endocrine responsive post-menopausal ER+ breast cancers.

Characterizing and exploiting the many roles of aberrant H2B monoubiquitination in cancer pathogenesis.

Monoubiquitination of histone H2B on lysine 120 (H2Bub1) is implicated in the control of multiple essential processes, including transcription, DNA damage repair and mitotic chromosome segregation. Accordingly, aberrant regulation of H2Bub1 can induce transcriptional reprogramming and genome instability that may promote oncogenesis. Remarkably, alterations of the ubiquitin ligases and deubiquitinating enzymes regulating H2Bub1 are emerging as ubiquitous features in cancer, further supporting the possibility that the misregulation of H2Bub1 is an underlying mechanism contributing to cancer pathogenesis. To date, aberrant H2Bub1 dynamics have been reported in multiple cancer types and are associated with transcriptional changes that promote oncogenesis in a cancer type-specific manner. Owing to the multi-functional nature of H2Bub1, misregulation of its writers and erasers may drive disease initiation and progression through additional synergistic processes. Accordingly, understanding the molecular determinants and pathogenic impacts associated with aberrant H2Bub1 regulation may reveal novel drug targets and therapeutic vulnerabilities that can be exploited to develop innovative precision medicine strategies that better combat cancer. In this review, we present the normal functions of H2Bub1 in the control of DNA-associated processes and describe the pathogenic implications associated with its misregulation in cancer. We further discuss the challenges coupled with the development of therapeutic strategies targeting H2Bub1 misregulation and expose the potential benefits of designing treatments that synergistically exploit the multiple functionalities of H2Bub1 to improve treatment selectivity and efficacy.

The F-box protein, FBXO7, is required to maintain chromosome stability in humans.

Despite the high morbidity and mortality rates associated with colorectal cancer (CRC), the aberrant genes and mechanisms driving CRC pathogenesis remain poorly understood. Chromosome instability (CIN), or ongoing changes in chromosome numbers, is a predominant form of genome instability associated with ~85% of CRCs, suggesting it may be a key mechanism driving CRC oncogenesis. CIN enables the acquisition of copy number alterations conferring selective growth, proliferation and survival advantages that promote cellular transformation. Despite these associations, the aberrant genes underlying CIN remain largely unknown. Candidate CIN gene FBXO7 encodes an F-box protein, a subunit of the SKP1-CUL1-FBOX (SCF) complex that confers substrate specificity to the complex and targets proteins for subsequent degradation by the 26S proteasome. Recently, the genes encoding the three core SCF complex members were identified as CIN genes; however, it is unknown whether F-box proteins exhibit similar integral roles in maintaining chromosome stability. Using short- small interfering RNA (siRNA) and long- (CRISPR/Cas9) term approaches, we show that reduced FBXO7 expression induces CIN in various colonic epithelial cell contexts, whereas FBXO7 knockout clones also exhibit hallmarks associated with cellular transformation, namely increased clonogenic and anchorage-independent growth. Collectively, these data demonstrate that FBXO7 is required to maintain genome stability identifying FBXO7 a novel CIN gene whose reduced expression may contribute to CRC development and progression.

Employing Cross-Species Approaches to Construct Humanized Genetic Interaction Networks.

Characterizing genetic interactions in humans, including synthetic lethal interactions, can provide fundamental insight into protein functions and pathway interactions. However, it can also assist in the development of innovative therapeutic strategies by uncovering novel drug targets used to combat diseases like cancer. To expedite the discovery of novel synthetic lethal interactions in humans, cross-species candidate gene approaches rely on the evolutionary conservation of genetic interactions between organisms. Here, we provide a guide that couples bioinformatic approaches and publicly available datasets from model organisms with cross-species approaches to expedite the identification of candidate synthetic lethal interactions to test in humans. First, we detail a method to identify relevant genetic interactions in budding yeast and subsequently provide a prioritization scheme to identify the most promising yeast interactions to pursue. Finally, we provide details on the tools and approaches used to identify the corresponding human orthologs to ultimately generate a testable network of candidate human synthetic lethal interactions. In summary, this approach leverages publicly available resources and datasets to expedite the identification of conserved synthetic lethal interactions in humans.

Exploring Candidate Human Synthetic Lethal Interactions Through siRNA and Quantitative Imaging-Based Approaches.

Synthetic lethal interactions can assist in characterizing protein functions and cellular processes, but they can also be used to identify novel drug targets for the development of innovative cancer therapeutic strategies. Despite recent technological advancements including CRISPR/Cas9 approaches, the systematic assessment of all pairwise gene interactions in humans (~ 200 million pairs) remains an unmet goal. Thus, hypothesis-driven approaches, which prioritize subsets of promising candidate SL interactions for experimental assessment, are critical to expedite the identification of novel SL interactions. Here, we provide a guide to screen and validate focused libraries of promising candidate SL interactions, typically consisting of 50-500 targets. First, we describe two siRNA and image-based screening protocols to rapidly assess candidate SL interactions. Subsequently, we provide methods to validate a subset of the most promising interactions uncovered in the screens. These approaches employ commercially available reagents and standard laboratory equipment to facilitate and expedite the identification of bona fide human SL interactions.

The SCF Complex Is Essential to Maintain Genome and Chromosome Stability.

The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.

Reduced USP22 Expression Impairs Mitotic Removal of H2B Monoubiquitination, Alters Chromatin Compaction and Induces Chromosome Instability That May Promote Oncogenesis.

Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.

Chromosome instability is prevalent and dynamic in high-grade serous ovarian cancer patient samples.

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) is the most lethal gynaecological malignancy in women with a high level of mortality, metastatic disease, disease recurrence and multi-drug resistance. Many previous studies have focused on characterising genome instability in recurrent resistant HGSOC and while this has advanced our understanding of HGSOC, our fundamental knowledge of the mechanisms driving genome instability remains limited. Chromosome instability (CIN; an increased rate of chromosome gains and losses) is a form of genome instability that is commonly associated with recurrence and multi-drug resistance in many cancer types but has just begun to be characterised in HGSOC. METHOD: To examine the relationship between CIN and HGSOC, we employed single-cell quantitative imaging microscopy approaches capable of capturing the cell-to-cell heterogeneity associated with CIN, to assess the prevalence and dynamics of CIN within individual and patient-matched HGSOC ascites and solid tumour samples. RESULTS: CIN occurs in 90.9% of ascites samples and 100% of solid tumours, while in-depth analyses identified statistically significant temporal dynamics within the serial ascites samples. In general, aneuploidy and CIN increase with disease progression and frequently decrease following chemotherapy treatments in responsive disease. Finally, our work identified higher levels of CIN in solid tumours relative to ascites samples isolated from the same individual, which identifies a novel difference existing between solid tumours and ascites samples. CONCLUSIONS: Our findings provide novel insight into the relationship between CIN and HGSOC, and uncover a previously unknown relationship existing between CIN in solid tumours and metastatic disease (ascites).

RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis.

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3-regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes-platinum resistance and angiogenesis.

A Comprehensive Assessment of Genetic and Epigenetic Alterations Identifies Frequent Variations Impacting Six Prototypic SCF Complex Members.

The SKP1, CUL1, F-box protein (SCF) complex represents a family of 69 E3 ubiquitin ligases that poly-ubiquitinate protein substrates marking them for proteolytic degradation via the 26S proteasome. Established SCF complex targets include transcription factors, oncoproteins and tumor suppressors that modulate cell cycle activity and mitotic fidelity. Accordingly, genetic and epigenetic alterations involving SCF complex member genes are expected to adversely impact target regulation and contribute to disease etiology. To gain novel insight into cancer pathogenesis, we determined the prevalence of genetic and epigenetic alterations in six prototypic SCF complex member genes (SKP1, CUL1, RBX1, SKP2, FBXW7 and FBXO5) from patient datasets extracted from The Cancer Genome Atlas (TCGA). Collectively, ~45% of observed SCF complex member mutations are predicted to impact complex structure and/or function in 10 solid tumor types. In addition, the distribution of encoded alterations suggest SCF complex members may exhibit either tumor suppressor or oncogenic mutational profiles in a cancer type dependent manner. Further bioinformatic analyses reveal the potential functional implications of encoded alterations arising from missense mutations by examining predicted deleterious mutations with available crystal structures. The SCF complex also exhibits frequent copy number alterations in a variety of cancer types that generally correspond with mRNA expression levels. Finally, we note that SCF complex member genes are differentially methylated across cancer types, which may effectively phenocopy gene copy number alterations. Collectively, these data show that SCF complex member genes are frequently altered at the genetic and epigenetic levels in many cancer types, which will adversely impact the normal targeting and timely destruction of protein substrates, which may contribute to the development and progression of an extensive array of cancer types.

Reduced RBX1 expression induces chromosome instability and promotes cellular transformation in high-grade serous ovarian cancer precursor cells.

Despite high-grade serous ovarian cancer (HGSOC) being the most common and lethal gynecological cancer in women, the early etiological events driving disease development remain largely unknown. Emerging evidence now suggests that chromosome instability (CIN; ongoing changes in chromosome numbers) may play a central role in the development and progression of HGSOC. Importantly, genomic amplification of the Cyclin E1 gene (CCNE1) contributes to HGSOC pathogenesis in ~20% of patients, while Cyclin E1 overexpression induces CIN in model systems. Cyclin E1 levels are normally regulated by the SCF (SKP1-CUL1-FBOX) complex, an E3 ubiquitin ligase that includes RBX1 as a core component. Interestingly, RBX1 is heterozygously lost in ~80% of HGSOC cases and reduced expression corresponds with worse outcomes, suggesting it may be a pathogenic event. Using both short (siRNA) and long (CRISPR/Cas9) term approaches, we show that reduced RBX1 expression corresponds with significant increases in CIN phenotypes in fallopian tube secretory epithelial cells, a cellular precursor of HGSOC. Moreover, reduced RBX1 expression corresponds with increased Cyclin E1 levels and anchorage-independent growth. Collectively, these data identify RBX1 as a novel CIN gene with pathogenic implications for HGSOC.