Expression of cancer testis antigens in human BRCA-associated breast cancers: potential targets for immunoprevention?

Introduction

Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers.

Methods

Archived breast cancer tissues (n?=?26) as well as morphologically normal breast tissues (n?=?7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE.

Results

CT antigens were expressed in 16/26 (61.5%, 95% CI 43?C80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P?=?0.003).

Conclusions

We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.     

Mouse models for BRCA1 associated tumorigenesis: From fundamental insights to preclinical utility

Germline mutations in BRCA1 result in a significant predisposition for breast and ovarian cancer, with frequent LOH of the remaining wild type allele. Soon after the identification of BRCA1, several different knockout mice were generated to study its biological function in vivo. BRCA1, which is involved in DNA double-strand break (DSB) repair, appeared to be essential for embryonic proliferation and survival during mid-gestation. In contrast to human mutation carriers however, heterozygous mouse mutants did not show spontaneous cancer development. Therefore, a number of conditional mouse models were developed. While tumors of these mice show varying degrees of similarity with their human counterparts, two mouse models develop mammary tumors that lack expression of estrogen and progesterone receptors and ERBB2. This ‘triple negative’ signature is a characteristic feature of BRCA1-associated breast cancers, which can therefore not be treated with endocrine agents or ERBB2-targeting therapeutics. Promising drugs for treating BRCA1-mutated tumors include platinum compounds and PARP inhibitors, which are specifically toxic to DSB repair deficient cells. Although encouraging results have been reported, recent findings indicate that BRCA1/2 deficient ovarian tumors can escape from such targeted treatment by genetic reversion. This resistance mechanism might be studied in future mouse tumor models based on Brca1 truncating mutations mimicking defined human founder mutations.     

Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers

Purpose

This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs) and to poly(ADP-ribose) polymerase (PARP) inhibitors.

Methods

Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.

Results

Thirty five (22.2%) of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7%) of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%), and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%). In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined) were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined) were identified in the hereditary non-triple-negative group.

Conclusions

Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.     

ALDH1A1 Maintains Ovarian Cancer Stem Cell-Like Properties by Altered Regulation of Cell Cycle Checkpoint and DNA Repair Network Signaling

Objective

Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.

Methods

Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.

Results

ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDH^HIGH displayed significantly lower progression free survival than the patients with ALDH^LOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.

Conclusion

This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.     

Clinical use and mechanisms of resistance for PARP inhibitors in homologous recombination-deficient cancers

Cells are continuously subjected to DNA damaging agents. DNA damages are repaired by one of the many pathways guarding genomic integrity. When one or several DNA damage pathways are rendered inefficient, cells can accumulate mutations, which modify normal cellular pathways, favoring abnormal cell growth. This supports malignant transformation, which can occur when cells acquire resistance to cell cycle checkpoints, apoptosis, or growth inhibition signals. Mutations in genes involved in the repair of DNA double strand breaks (DSBs), such as BRCA1, BRCA2, or PALB2, significantly increase the risk of developing cancer of the breast, ovaries, pancreas, or prostate. Fortunately, the inability of these tumors to repair DNA breaks makes them sensitive to genotoxic chemotherapies, allowing for the development of therapies precisely tailored to individuals’ genetic backgrounds. Unfortunately, as with many anti-cancer agents, drugs used to treat patients carrying a BRCA1 or BRCA2 mutation create a selective pressure, and over time tumors can become drug resistant. Here, we detail the cellular function of tumor suppressors essential in DNA damage repair pathways, present the mechanisms of action of inhibitors used to create synthetic lethality in BRCA carriers, and review the major molecular sources of drug resistance. Finally, we present examples of the many strategies being developed to circumvent drug resistance.     

HspBP1 is a dual function regulatory protein that controls both DNA repair and apoptosis in breast cancer cells

The Hsp70-binding protein 1 (HspBP1) belongs to a family of co-chaperones that regulate Hsp70 activity and whose biological significance is not well understood. In the present study, we show that when HspBP1 is either knocked down or overexpressed in BRCA1-proficient breast cancer cells, there were profound changes in tumorigenesis, including anchorage-independent cell growth in vitro and in tumor formation in xenograft models. However, HspBP1 did not affect tumorigenic properties in BRCA1-deficient breast cancer cells. The mechanisms underlying HspBP1-induced tumor suppression were found to include interactions with BRCA1 and promotion of BRCA1-mediated homologous recombination DNA repair, suggesting that HspBP1 contributes to the suppression of breast cancer by regulating BRCA1 function and thereby maintaining genomic stability. Interestingly, independent of BRCA1 status, HspBP1 facilitates cell survival in response to ionizing radiation (IR) by interfering with the association of Hsp70 and apoptotic protease-activating factor-1. These findings suggest that decreased HspBP1 expression, a common occurrence in high-grade and metastatic breast cancers, leads to genomic instability and enables resistance to IR treatment.Subject terms: Homologous recombination, Breast cancer     

Functional profiling of nucleotide Excision repair in breast cancer

Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.     

Genomic rearrangements in BRCA1 and BRCA2: A literature review

Women with mutations in the breast cancer genes BRCA1 or BRCA2 have an increased lifetime risk of developing breast, ovarian and other BRCA-associated cancers. However, the number of detected germline mutations in families with hereditary breast and ovarian cancer (HBOC) syndrome is lower than expected based upon genetic linkage data. Undetected deleterious mutations in the BRCA genes in some high-risk families are due to the presence of intragenic rearrangements such as deletions, duplications or insertions that span whole exons. This article reviews the molecular aspects of BRCA1 and BRCA2 rearrangements and their frequency among different populations. An overview of the techniques used to screen for large rearrangements in BRCA1 and BRCA2 is also presented. The detection of rearrangements in BRCA genes, especially BRCA1, offers a promising outlook for mutation screening in clinical practice, particularly in HBOC families that test negative for a germline mutation assessed by traditional methods.     

Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

Cisplatin is a cytotoxic platinum compound that triggers DNA crosslinking induced cell death, and is one of the reference drugs used in the treatment of several types of human cancers including gastric cancer. However, intrinsic or acquired drug resistance to cisplatin is very common, and leading to treatment failure. We have recently shown that reduced expression of base excision repair protein XRCC1 (X-ray repair cross complementing group1) in gastric cancerous tissues correlates with a significant survival benefit from adjuvant first-line platinum-based chemotherapy. In this study, we demonstrated the role of XRCC1 in repair of cisplatin-induced DNA lesions and acquired cisplatin resistance in gastric cancer by using cisplatin-sensitive gastric cancer cell lines BGC823 and the cisplatin-resistant gastric cancer cell lines BGC823/cis-diamminedichloridoplatinum(II) (DDP). Our results indicated that the protein expression of XRCC1 was significantly increased in cisplatin-resistant cells and independently contributed to cisplatin resistance. Irinotecan, another chemotherapeutic agent to induce DNA damaging used to treat patients with advanced gastric cancer that progressed on cisplatin, was found to inhibit the expression of XRCC1 effectively, and leading to an increase in the sensitivity of resistant cells to cisplatin. Our proteomic studies further identified a cofactor of 26S proteasome, the thioredoxin-like protein 1 (TXNL1) that downregulated XRCC1 in BGC823/DDP cells via the ubiquitin-proteasome pathway. In conclusion, the TXNL1-XRCC1 is a novel regulatory pathway that has an independent role in cisplatin resistance, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.     

TRIM44 promotes BRCA1 functions in HR repair to induce Cisplatin Chemoresistance in Lung Adenocarcinoma by Deubiquitinating FLNA

Tripartite motif-containing 44 (TRIM44) has recently been implicated in various pathological processes in numerous cancers, including lung adenocarcinoma (LUAD); however, its functional roles in chemoresistance are poorly understood. Herein, TRIM44 knockdown sensitized LUAD cells to cisplatin and enhanced cisplatin-induced apoptosis. Microarray analysis indicated that the “Role of BRCA1 in DNA damage” and the BRCA1 gene expression were positively regulated by TRIM44, which was further verified by immunofluorescence, qRT-PCR, and Western blotting. BRCA1 depletion effectively abolished TRIM44-modulated cisplatin resistance and regulation of homologous recombination (HR) repair. Interestingly, TRIM44 interacted with FLNA, an upstream regulator of BRCA1 as specified by STRING V 11.5, and facilitated its stability and deubiquitination. FLNA was also found to be required for the functions of TRIM44 in drug resistance. Using animal models, overexpression of TRIM44 was shown to confer resistance to cisplatin in a BRCA1- and FLNA-dependent manner. TRIM44 expression levels in tissues from cisplatin-resistant LUAD patients were significantly higher than those in tissues from cisplatin-sensitive LUAD patients. Collectively, our study results demonstrate that the TRIM44/FLNA/BRCA1 axis is involved in cisplatin chemoresistance, providing potential therapeutic targets for LUAD patients with cisplatin resistance.     

PARP inhibitors in ovarian cancer: Sensitivity prediction and resistance mechanisms

Poly (ADP‐ribose) polymerase (PARP) inhibitors have provided great clinical benefits to ovarian cancer patients. To date, three PARP inhibitors, namely, olaparib, rucaparib and niraparib have been approved for the treatment of ovarian cancer in the United States. Homologous recombination deficiency (HRD) and platinum sensitivity are prospective biomarkers for predicting the response to PARP inhibitors in ovarian cancers. Preclinical data have focused on identifying the gene aberrations that might generate HRD and induce sensitivity to PARP inhibitors in vitro in cancer cell lines or in vivo in patient‐derived xenografts. Clinical trials have focused on genomic scar analysis to identify biomarkers for predicting the response to PARP inhibitors. Additionally, researchers have aimed to investigate mechanisms of resistance to PARP inhibitors and strategies to overcome this resistance. Combining PARP inhibitors with HR pathway inhibitors to extend the utility of PARP inhibitors to BRCA‐proficient tumours is increasingly foreseeable. Identifying the population of patients with the greatest potential benefit from PARP inhibitor therapy and the circumstances under which patients are no longer suited for PARP inhibitor therapy are important. Further studies are required in order to propose better strategies for overcoming resistance to PARP inhibitor therapy in ovarian cancers.     

A Radiation Hybrid Map of the BRCA1 Region

A locus on chromosome 17q, designated “BRCA1,” has been identified as a predisposition gene for breast cancer. A panel of chromosome 17–specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation     

Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would “fuel” enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immunostaining and quantitation of vessel density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by overexpression of PGC-1β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial “power” in epithelial cancer cells oncogenically promotes tumor growth by conferring autophagy resistance. As such, PGC-1α, PGC-1β, mitoNEET and POLRMT should all be considered as tumor promoters or “metabolic oncogenes.” Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial “poison”) prevents the onset of nearly all types of human cancers in diabetic patients. Therefore, metformin (a complex I inhibitor) and other mitochondrial inhibitors should be developed as novel anticancer therapies, targeting mitochondrial metabolism in cancer cells.