Functional Analysis of Familial Asp67Glu and Thr1051Ser BRCA1 Mutations in Breast/Ovarian Carcinogenesis

Estrogen is believed to be pre-initiator in the risk of breast cancer. The BRCA1 is a tumor suppressor gene associated with breast and ovarian cancer risk. This report describes functional analysis of two BRCA1 missense mutations (Asp67Glu and Thr1051Ser) observed in the familial breast/ovarian cancer patients in Thailand. Levels of luciferase activity of the two mutations were relatively lower than in the wild-type BRCA1. It is indicated that mutants may fail to promote the estrogen receptor dependent functions. It is presumed that estrogen and insulin/IGF-1 regulate c-Myc and cyclin D1 during breast cancer cell proliferation. It is also likely to affect ubiquitination mechanism. Since three affected cancer families carry the Asp67Glu mutation, it is believed that this type of mutation could have some effect on breast/ovarian cancer progression.     

Breast Cancer Predisposition Genes and Synthetic Lethality

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.     

Ovarian Cancer: In Search of Better Marker Systems Based on DNA Repair Defects

Ovarian cancer is the fifth most common female cancer in the Western world, and the deadliest gynecological malignancy. The overall poor prognosis for ovarian cancer patients is a consequence of aggressive biological behavior and a lack of adequate diagnostic tools for early detection. In fact, approximately 70% of all patients with epithelial ovarian cancer are diagnosed at advanced tumor stages. These facts highlight a significant clinical need for reliable and accurate detection methods for ovarian cancer, especially for patients at high risk. Because CA125 has not achieved satisfactory sensitivity and specificity in detecting ovarian cancer, numerous efforts, including those based on single and combined molecule detection and “omics” approaches, have been made to identify new biomarkers. Intriguingly, more than 10% of all ovarian cancer cases are of familial origin. BRCA1 and BRCA2 germline mutations are the most common genetic defects underlying hereditary ovarian cancer, which is why ovarian cancer risk assessment in developed countries, aside from pedigree analysis, relies on genetic testing of BRCA1 and BRCA2. Because not only BRCA1 and BRCA2 but also other susceptibility genes are tightly linked with ovarian cancer-specific DNA repair defects, another possible approach for defining susceptibility might be patient cell-based functional testing, a concept for which support came from a recent case-control study. This principle would be applicable to risk assessment and the prediction of responsiveness to conventional regimens involving platinum-based drugs and targeted therapies involving poly (ADP-ribose) polymerase (PARP) inhibitors.     

Sanger Sequencing for BRCA1 c.68_69del,BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.     

Genetic Aberrations of DNA Repair Pathways in Prostate Cancer: Translation to the Clinic

Prostate cancer (PC) is the second most common cancer in men worldwide. Due to the large-scale sequencing efforts, there is currently a better understanding of the genomic landscape of PC. The identification of defects in DNA repair genes has led to clinical studies that provide a strong rationale for developing poly (ADP-ribose) polymerase (PARP) inhibitors and DNA-damaging agents in this molecularly defined subset of patients. The identification of molecularly defined subgroups of patients has also other clinical implications; for example, we now know that carriers of breast cancer 2 (BRCA2) pathogenic sequence variants (PSVs) have increased levels of serum prostate specific antigen (PSA) at diagnosis, increased proportion of high Gleason tumors, elevated rates of nodal and distant metastases, and high recurrence rate; BRCA2 PSVs confer lower overall survival (OS). Distinct tumor PSV, methylation, and expression patterns have been identified in BRCA2 compared with non-BRCA2 mutant prostate tumors. Several DNA damage response and repair (DDR)-targeting agents are currently being evaluated either as single agents or in combination in patients with PC. In this review article, we highlight the biology and clinical implications of deleterious inherited or acquired DNA repair pathway aberrations in PC and offer an overview of new agents being developed for the treatment of PC.     

PARP Inhibitors Display Differential Efficacy in Models of BRCA Mutant High-Grade Serous Ovarian Cancer

Several poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors are now in clinical use for tumours with defects in BReast CAncer genes BRCA1 or BRCA2 that result in deficient homologous recombination repair (HRR). Use of olaparib, niraparib or rucaparib for the treatment of high-grade serous ovarian cancer, including in the maintenance setting, has extended both progression free and overall survival for women with this malignancy. While different PARP inhibitors (PARPis) are mechanistically similar, differences are apparent in their chemical structures, toxicity profiles, PARP trapping abilities and polypharmacological landscapes. We have treated ovarian cancer cell line models of known BRCA status, including the paired cell lines PEO1 and PEO4, and UWB1.289 and UWB1.289+BRCA1, with five PARPis (olaparib, niraparib, rucaparib, talazoparib and veliparib) and observed differences between PARPis in both cell viability and cell survival. A cell line model of acquired resistance to veliparib showed increased resistance to the other four PARPis tested, suggesting that acquired resistance to one PARPi may not be able to be rescued by another. Lastly, as a proof of principle, HRR proficient ovarian cancer cells were sensitised to PARPis by depletion of BRCA1. In the future, guidelines will need to emerge to assist clinicians in matching specific PARPis to specific patients and tumours.     

Hereditary Prostate Cancer: Genes Related,Target Therapy and Prevention

Prostate cancer (PCa) is globally the second most diagnosed cancer type and the most common cause of cancer-related deaths in men. Family history of PCa, hereditary breast and ovarian cancer (HBOC) and Lynch syndromes (LS), are among the most important risk factors compared to age, race, ethnicity and environmental factors for PCa development. Hereditary prostate cancer (HPCa) has the highest heritability of any major cancer in men. The proportion of PCa attributable to hereditary factors has been estimated in the range of 5–15%. To date, the genes more consistently associated to HPCa susceptibility include mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) and homologous recombination genes (BRCA1/2, ATM, PALB2, CHEK2). Additional genes are also recommended to be integrated into specific research, including HOXB13, BRP1 and NSB1. Importantly, BRCA1/BRCA2 and ATM mutated patients potentially benefit from Poly (ADP-ribose) polymerase PARP inhibitors, through a mechanism of synthetic lethality, causing selective tumor cell cytotoxicity in cell lines. Moreover, the detection of germline alterations in MMR genes has therapeutic implications, as it may help to predict immunotherapy benefits. Here, we discuss the current knowledge of the genetic basis for inherited predisposition to PCa, the potential target therapy, and the role of active surveillance as a management strategy for patients with low-risk PCa. Finally, the current PCa guideline recommendations are reviewed.     

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.     

HE4 in the Differential Diagnosis of a Pelvic Mass: A Case Report

Neoplasms of the ovary present an increasing challenge to the physician. Neoplastic ovarian cysts can resemble endometriomas in ultrasound imaging and need to be carefully considered in the differential diagnosis. We report the case of a woman with a strong family history of hereditary breast and ovarian cancer, who presented with a pelvic mass. The young girl refused oncogenetic counseling and genetic testing, even though she had a 50% a priori probability of being a BRCA1 mutation carrier. Pelvic magnetic resonance imaging (MRI) and a comparative analysis of the serum concentration of HE-4 and CA125 biomarkers provided accuracy and sensitivity in the diagnosis of a benign ovarian pathology. Based on this experience, we propose that the sensitivity of a screening program based on a HE4 and CA125 assay and MRI in high risk patients with mutations in the BRCA1 and BRCA2 genes may be considered a useful pre-operative tool for the differential diagnosis of pelvic masses.     

Chimeric Antigen Receptor Design and Efficacy in Ovarian Cancer Treatment

Our increased understanding of tumour biology gained over the last few years has led to the development of targeted molecular therapies, e.g., vascular endothelial growth factor A (VEGF-A) antagonists, poly[ADP-ribose] polymerase 1 (PARP1) inhibitors in hereditary breast and ovarian cancer syndrome (BRCA1 and BRCA2 mutants), increasing survival and improving the quality of life. However, the majority of ovarian cancer (OC) patients still do not have access to targeted molecular therapies that would be capable of controlling their disease, especially resistant or relapsed. Chimeric antigen receptors (CARs) are recombinant receptor constructs located on T lymphocytes or other immune cells that change its specificity and functions. Therefore, in a search for a successful solid tumour therapy using CARs the specific cell surface antigens identification is crucial. Numerous in vitro and in vivo studies, as well as studies on humans, prove that targeting overexpressed molecules, such as mucin 16 (MUC16), annexin 2 (ANXA2), receptor tyrosine-protein kinase erbB-2 (HER2/neu) causes high tumour cells toxicity and decreased tumour burden. CARs are well tolerated, side effects are minimal and they inhibit disease progression. However, as OC is heterogenic in its nature with high mutation diversity and overexpression of different receptors, there is a need to consider an individual approach to treat this type of cancer. In this publication, we would like to present the history and status of therapies involving the CAR T cells in treatment of OC tumours, suggest potential T cell-intrinsic determinants of response and resistance as well as present extrinsic factors impacting the success of this approach.     

The Correlation of Mutations and Expressions of Genes within the PI3K/Akt/mTOR Pathway in Breast Cancer—A Preliminary Study

There is an urgent need to seek new molecular biomarkers helpful in diagnosing and treating breast cancer. In this elaboration, we performed a molecular analysis of mutations and expression of genes within the PI3K/Akt/mTOR pathway in patients with ductal breast cancer of various malignancy levels. We recognized significant correlations between the expression levels of the studied genes. We also performed a bioinformatics analysis of the data available on the international database TCGA and compared them with our own research. Studies on mutations and expression of genes were conducted using High-Resolution Melt PCR (HRM-PCR), Allele-Specific-quantitative PCR (ASP-qPCR), Real-Time PCR molecular methods in a group of women with ductal breast cancer. Bioinformatics analysis was carried out using web source Ualcan and bc-GenExMiner. In the studied group of women, it was observed that the prevalence of mutations in the studied PIK3CA and AKT1 genes was 29.63%. It was stated that the average expression level of the PIK3CA, PIK3R1, PTEN genes in the group of breast cancer patients is lower in comparison to the control group, while the average expression level of the AKT1 and mTOR genes in the studied group was higher in comparison to the control group. It was also indicated that in the group of patients with mutations in the area of the PIK3CA and AKT1 genes, the PIK3CA gene expression level is statistically significantly lower than in the group without mutations. According to our knowledge, we demonstrate, for the first time, that there is a very strong positive correlation between the levels of AKT1 and mTOR gene expression in the case of patients with mutations and without mutations.     

Moving beyond PARP Inhibition: Current State and Future Perspectives in Breast Cancer

Breast cancer is the most frequent and lethal tumor in women and finding the best therapeutic strategy for each patient is an important challenge. PARP inhibitors (PARPis) are the first, clinically approved drugs designed to exploit synthetic lethality in tumors harboring BRCA1/2 mutations. Recent evidence indicates that PARPis have the potential to be used both in monotherapy and combination strategies in breast cancer treatment. In this review, we show the mechanism of action of PARPis and discuss the latest clinical applications in different breast cancer treatment settings, including the use as neoadjuvant and adjuvant approaches. Furthermore, as a class, PARPis show many similarities but also certain critical differences which can have essential clinical implications. Finally, we report the current knowledge about the resistance mechanisms to PARPis. A systematic PubMed search, using the entry terms “PARP inhibitors” and “breast cancer”, was performed to identify all published clinical trials (Phase I-II-III) and ongoing trials (ClinicalTrials.gov), that have been reported and discussed in this review.     

Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(��6-p-phenylethacrynate)Cl2(pta)]

The ruthenium-based complex [Ru(η⁶-p-phenylethacrynate)Cl₂(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane), termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1) of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.     

The Antitumor Effect of Metformin Is Mediated by miR-26a in Breast Cancer

Metformin, a drug approved for diabetes type II treatment, has been associated with a reduction in the incidence of breast cancer and metastasis and increased survival in diabetic breast cancer patients. High levels of miR-26a expression have been proposed as one of the possible mechanisms for this effect; likewise, this miRNA has also been associated with survival/apoptosis processes in breast cancer. Our aim was to evaluate if miR-26a and some of its targets could mediate the effect of metformin in breast cancer. The viability of MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cell lines was evaluated with an MTT assay after ectopic overexpression and/or downregulation of miR-26a. Similarly, the expression levels of the miR-26a targets CASP3, CCNE2, ABL2, APAF1, XIAP, BCL-2, PTEN, p53, E2F3, CDC25A, BCL2L1, MCL-1, EZH2, and MTDH were assessed by quantitative polymerase chain reaction (PCR). The effect of metformin treatment on breast cancer cell viability and miR-26a, BCL-2, PTEN, MCL-1, EZH2, and MTDH modulation were evaluated. Wound healing experiments were performed to analyze the effect of miR-26a and metformin treatment on cell migration. MiR-26a overexpression resulted in a reduction in cell viability that was partially recovered by inhibiting it. E2F3, MCL-1, EZH2, MTDH, and PTEN were downregulated by miR-26a and the PTEN (phosphatase and tensin homolog) protein was also reduced after miR-26a overexpression. Metformin treatment reduced breast cancer cell viability, increased miR-26a expression, and led to a reduction in BCL-2, EZH2, and PTEN expression. miR-26a inhibition partly prevents the metformin viability effect and the PTEN and EZH2 expression reduction. Our results indicate that metformin effectively reduces breast cancer cell viability and suggests that the effects of the drug are mediated by an increase in miR-26a expression and a reduction of its targets, PTEN and EHZ2 Thus, the use of metformin in breast cancer treatment constitutes a promising potential breast cancer therapy.     

Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.     

Inherited Retinal Diseases Due to RPE65 Variants: From Genetic Diagnostic Management to Therapy

Inherited retinal diseases (IRDs) are a heterogeneous group of conditions that include retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EO[S]RD), which differ in severity and age of onset. IRDs are caused by mutations in >250 genes. Variants in the RPE65 gene account for 0.6–6% of RP and 3–16% of LCA/EORD cases. Voretigene neparvovec is a gene therapy approved for the treatment of patients with an autosomal recessive retinal dystrophy due to confirmed biallelic RPE65 variants (RPE65-IRDs). Therefore, the accurate molecular diagnosis of RPE65-IRDs is crucial to identify ‘actionable’ genotypes—i.e., genotypes that may benefit from the treatment—and is an integral part of patient management. To date, hundreds of RPE65 variants have been identified, some of which are classified as pathogenic or likely pathogenic, while the significance of others is yet to be established. In this review, we provide an overview of the genetic diagnostic workup needed to select patients that could be eligible for voretigene neparvovec treatment. Careful clinical characterization of patients by multidisciplinary teams of experts, combined with the availability of next-generation sequencing approaches, can accelerate patients’ access to available therapeutic options.     

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.     

Association of the rs1346044 Polymorphism of the Werner Syndrome Gene RECQL2 with Increased Risk and Premature Onset of Breast Cancer

Like other RECQ helicases, WRN/RECQL2 plays a crucial role in DNA replication and the maintenance of genome stability. Inactivating mutations in RECQL2 lead to Werner syndrome, a rare autosomal disease associated with premature aging and an increased susceptibility to multiple cancer types. We analyzed the association of two coding single-nucleotide polymorphisms in WRN, Cys1367Arg (rs1346044), and Arg834Cys (rs3087425), with the risk, age at onset, and clinical subclasses of breast cancer in a hospital-based case-control study of an Austrian population of 272 breast cancer patients and 254 controls. Here we report that the rare homozygous CC genotype of rs1346044 was associated with an approximately two-fold elevated breast cancer risk. Moreover, patients with the CC genotype exhibited a significantly increased risk of developing breast cancer under the age of 55 in both recessive and log-additive genetic models. CC patients developed breast cancer at a mean age of 55.2 ± 13.3 years and TT patients at 60.2 ± 14.7 years. Consistently, the risk of breast cancer was increased in pre-menopausal patients in the recessive model. These findings suggest that the CC genotype of WRN rs1346044 may contribute to an increased risk and a premature onset of breast cancer.     

The Role of Sialyl-Tn in Cancer

Activation of an aberrant glycosylation pathway in cancer cells can lead to expression of the onco-foetal sialyl-Tn (sTn) antigen. STn is a truncated O-glycan containing a sialic acid α-2,6 linked to GalNAc α-O-Ser/Thr and is associated with an adverse outcome and poor prognosis in cancer patients. The biosynthesis of the sTn antigen has been linked to the expression of the sialytransferase ST6GalNAc1, and also to mutations in and loss of heterozygosity of the COSMC gene. sTn neo- or over-expression occurs in many types of epithelial cancer including gastric, colon, breast, lung, oesophageal, prostate and endometrial cancer. sTn is believed to be carried by a variety of glycoproteins and may influence protein function and be involved in tumour development. This review discusses how the role of sTn in cancer development and tumour cell invasiveness might be organ specific and occur through different mechanisms depending on each cancer type or subtype. As the sTn-antigen is expressed early in carcinogenesis targeting sTn in cancer may enable the targeting of tumours from the earliest stage.