Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair

Background:

In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair.

Methods:

Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay.

Results:

Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency.

Conclusion:

These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.     

Tumour-suppressive function of SIRT4 in human colorectal cancer

Background:

SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers.

Methods:

We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry.

Results:

SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis.

Conclusions:

SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.     

Association between cytosolic expression of BRCA1 and metastatic risk in breast cancer

Background:

Although BRCA1 has been extensively studied for its role as a tumour-suppressor protein, the role of BRCA1 subcellular localisation in oncogenesis and tumour progression has remained unclear. This study explores the impact of BRCA1 mislocalisation on clinical outcomes in breast cancer.

Methods:

Tissue microarrays assembled from a cohort of patients with all stages of breast cancer were analysed for BRCA1 localisation and correlated with patient survival. Tissue microarrays of patients who had breast cancer that had metastasised to the lung were assembled from an independent cohort of patients. These were analysed for BRCA1 subcellular expression. In vitro studies using cultured human breast cancer cells were conducted to examine the effect of cytosolic BRCA1 on cell migration and efficiency of invasion.

Results:

An inverse association was found between cytosolic BRCA1 expression and metastasis-free survival in patients aged >40 years. Further analysis of BRCA1 subcellular expression in a cohort of breast cancer patients with metastatic disease revealed that the cytosolic BRCA1 content of breast tumours that had metastasised to the lung was 36.0% (95% CI=(31.7%, 40.3%), which was markedly higher than what is reported in the literature (8.2–14.8%). Intriguingly, these lung metastases and their corresponding primary breast tumours demonstrated similarly high cytosolic BRCA1 distributions in both paired and unpaired analyses. Finally, in vitro studies using human breast cancer cells demonstrated that genetically induced BRCA1 cytosolic sequestration (achieved using the cytosol-sequestering BRCA1 5382insC mutation) increased cell invasion efficiency.

Conclusions:

Results from this study suggest a model where BRCA1 cytosolic mislocalisation promotes breast cancer metastasis, making it a potential biomarker of metastatic disease.     

BRCA1 haplotype and clinical benefit of trabectedin in soft-tissue sarcoma patients

Background:

This study aimed to determine whether the BRCA1 haplotype was associated with trabectedin efficacy in soft-tissue sarcoma (STS) patients.

Methods:

We analysed BRCA1 single-nucleotide polymorphisms (SNPs) in tumour specimens from 135 advanced STS patients enrolled in published phase 2 trials or in a compassionate-use programme of trabectedin. Forty-four advanced STS patients treated with doxorubicin and 85 patients with localised STS served as controls. The 6-month nonprogression rate and overall survival (OS) were analysed according to BRCA1 haplotype using log-rank tests.

Results:

A favourable BRCA1 haplotype (presence of at least one AAAG allele) was significantly associated with an improved 6-month nonprogression rate. It was the only variable significantly associated with OS. No correlations were found between outcomes for patients with localised or advanced STS treated with doxorubicin.

Conclusions:

The BRCA1 haplotype represents a potential DNA repair biomarker that can be used for the prediction of response to trabectedin in STS patients.     

BRCA1 and BRCA2 mutations sensitize to chemotherapy in patient-derived pancreatic cancer xenografts

Background:

Germline mutations of the BRCA tumour suppressors have been associated with increased risk of pancreatic cancer. Clinical evidence suggests that these patients may be more sensitive to treatment with cisplatin. As the frequency of germline BRCA mutations is low, definitive experimental data to support the clinical observations are still missing.

Methods:

We tested gemcitabine and cisplatin sensitivity of four BRCA1 and BRCA2 mutant and three BRCA1 and BRCA2 wild-type (WT) patient-derived pancreatic cancer xenografts.

Results:

We observed treatment sensitivity to gemcitabine and cisplatin in the BRCA WT and mutant models. The BRCA1 and BRCA2 mutant xenografts were significantly more sensitive to cisplatin although these models also showed sensitivity to gemcitabine. The BRCA1 and BRCA2 WT models showed sensitivity to gemcitabine but not cisplatin. Treatment sensitivity in the xenograft models closely resembled treatment response in the corresponding patients.

Discussion:

We have characterised a panel of xenografts derived from pancreatic cancer patients carrying germline BRCA mutations, and shown that their genetic features resemble the patient donor. Our results support further clinical testing of treatment regimens combining gemcitabine and platinum drugs in this patient population, as well as preclinical research aiming to identify mechanisms of cisplatin resistance in BRCA mutant pancreatic cancers.     

Low-level constitutional mosaicism of a de novoBRCA1 gene mutation

Background:

Pathogenic BRCA1 mutations are usually inherited. Constitutional low-level BRCA1 mosaicism has never been reported.

Methods:

Next-generation sequencing (NGS) of cancer gene panel of germline and tumour DNA in a patient with early onset, triple-negative breast cancer.

Results:

Constitutional de novo mosaicism (5%) for a pathogenic (c.1953dupG; p.Lys652Glufs*21) BRCA1mutation was detected in leukocytes, buccal tissue and normal breast tissue DNA, with ∼50% mutation in tumorous breast tissue.

Conclusion:

This is the first reported case of low-level, multiple tissue, constitutional mosaicism in BRCA1, and highlights the need to consider deep sequencing in affected individuals clinically suspected of having cancer predisposition whose tumours display a BRCA mutation.     

Absence of truncating BRIP1 mutations in chromosome 17q-linked hereditary prostate cancer families

Background:

In a genome-wide scan (GWS) of 175 multiplex prostate cancer (PCa) families from the University of Michigan Prostate Cancer Genetics Project (PCGP), linkage was observed to markers on chromosome 17q21–24, a region that includes two breast cancer susceptibility genes, BRCA1 and BRIP1. BRIP1 is a Fanconi anaemia gene (FANCJ) that interacts with the BRCT domain of BRCA1 and has a role in DNA damage repair. Protein truncating mutations in BRIP1 have been identified in hereditary breast and ovarian cancer families, and a recent report suggested that a recurrent truncating mutation (R798X) may have a role in PCa susceptibility.

Methods:

We examined the role of BRIP1 mutations in hereditary PCa through sequence analysis of 94 individuals from PCGP families showing linkage to 17q.

Results:

A total of 24 single-nucleotide polymorphisms, including 7 missense variants but no protein truncating mutations, were observed.

Conclusion:

The data presented here suggest that BRIP1 truncating mutations are uncommon in PCa cases and do not account for the linkage to chromosome 17q observed in our GWS. Additional investigation is needed to determine the significance, if any, of the observed BRIP1 missense variants in hereditary PCa.     

Expression of oncogenic BARD1 isoforms affects colon cancer progression and correlates with clinical outcome

Background:

Colon cancer predisposition is associated with mutations in BRCA1. BRCA1 protein stability depends on binding to BARD1. In different cancers, expression of differentially spliced BARD1 isoforms is correlated with poor prognosis and decreased patient survival. We therefore suspected a role of BARD1 isoforms in colon cancer.

Methods:

We performed immunohistochemistry in 168 colorectal cancers, using four antibodies directed against differentially expressed regions of BARD1. We determined structure and relative expression of BARD1 mRNA isoforms in 40 tumour and paired normal peri-tumour tissues. BARD1 expression was correlated with clinical outcome.

Results:

BARD1 isoforms were expressed in 98% of cases and not correlated with BRCA1. BARD1 mRNA isoforms were upregulated in all tumours as compared with paired normal peri-tumour tissues. Non-correlated expression and localisation of different epitopes suggested insignificant expression of full-length (FL) BARD1. Expression of N- and C-terminal epitopes correlated with increased survival, but expression of epitopes mapping to the middle of BARD1 correlated with decreased survival. Middle epitopes are present in oncogenic BARD1 isoforms, which have pro-proliferative functions. Correlated upregulation of only N- and C-terminal epitopes reflects the expression of isoforms BARD1δ and BARD1ϕ.

Conclusion:

Our results suggest that BARD1 isoforms, but not FL BARD1, are expressed in colon cancer and affect its progression and clinical outcome.     

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA)

Background:

In this study we aimed to evaluate the role of a SNP in intron 1 of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers.

Methods:

We have genotyped rs744154 in 9408 BRCA1 and 5632 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and assessed its association with breast cancer risk using a retrospective weighted cohort approach.

Results:

We found no evidence of association with breast cancer risk for BRCA1 (per-allele HR: 0.98, 95% CI: 0.93–1.04, P=0.5) or BRCA2 (per-allele HR: 0.97, 95% CI: 0.89–1.06, P=0.5) mutation carriers.

Conclusion:

This SNP is not a significant modifier of breast cancer risk for mutation carriers, though weak associations cannot be ruled out.     

MicroRNA-based molecular classification of non-BRCA1/2 hereditary breast tumours

Background:

Hereditary breast cancer comprises 5–10% of all breast cancers. Mutations in two high-risk susceptibility genes, BRCA1 and BRCA2, along with rare intermediate-risk genes and common low-penetrance alleles identified, altogether explain no more than 45% of the high-risk breast cancer families, although the majority of cases are unaccounted for and are designated as BRCAX tumours. Micro RNAs have called great attention for classification of different cancer types and have been implicated in a range of important biological processes and are deregulated in cancer pathogenesis.

Methods:

Here we have performed an exploratory hypothesis-generating study of miRNA expression profiles in a large series of 66 primary hereditary breast tumours by microarray analysis.

Results:

Unsupervised clustering analysis of miRNA molecular profiles revealed distinct subgroups of BRCAX tumours, ‘normal-like'' BRCAX-A, ‘proliferative'' BRCAX-B, ‘BRCA1/2-like'' BRCAX-C and ‘undefined'' BRCAX-D subgroup. These findings introduce a new insight in the biology of hereditary breast cancer, defining specific BRCAX subgroups, which could help in the search for novel susceptibility pathways in hereditary breast cancer.

Conclusion:

Our data demonstrate that BRCAX hereditary breast tumours can be sub-classified into four previously unknown homogenous groups characterised by specific miRNA expression signatures and histopathological features.     

A comparison of the detection of BRCA mutation carriers through the provision of Jewish population-based genetic testing compared with clinic-based genetic testing

Background:

Guidelines for genetic testing for BRCA1 or BRCA2 stipulate that a personal or family history of cancer is necessary to be eligible for testing. Approximately 2% of Ashkenazi Jewish women carry a mutation, but to date population-based testing has not been advocated. Little is known about the relative yield of a conventional genetic testing programme versus a programme of widespread testing in a population with common founder mutations.

Methods:

We provided both referral-based and Jewish population-based testing between 2008 and 2012. We compared the numbers of BRCA mutation carriers identified through the two streams and estimated the number of genetic counselling hours devoted to each programme.

Results:

From 2008 to 2012, 38 female carriers were identified through 487 referrals to our genetics centre (29 unaffected with cancer). During the same time, 6179 Jewish women were tested through our population-based programme and 93 mutation carriers were identified (92 unaffected with cancer). Fewer counsellor hours were devoted to the population-based than to the clinical referral-based testing programme.

Conclusion:

Genetic testing of all Jewish women above the age of 25 years will greatly expand the number of BRCA mutation carriers identified without a commensurate increase in the number of hours required for counselling.     

Tailoring heated intraperitoneal mitomycin C for peritoneal metastases originating from colorectal carcinoma: a translational approach to improve survival

Background:

Patients with peritoneal metastases (PMs) originating from colorectal carcinoma (CRC) are curatively treated by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC). We aim to improve patient selection for HIPEC by predicting MMC sensitivity.

Methods:

The MMC sensitivity was determined for 12 CRC cell lines and correlated to mRNA expression of 37 genes related to the Fanconi anaemia (FA)–BRCA pathway, ATM–ATR pathway and enzymatic activation of MMC. Functionality of the FA–BRCA pathway in cell lines was assessed using a chromosomal breakage assay and western blot for key protein FANCD2. Bloom syndrome protein (BLM) was further analysed by staining for the corresponding protein with immunohistochemistry (IHC) on both CRC cell lines (n=12) and patient material (n=20).

Results:

High sensitivity correlated with a low BLM (P=0.01) and BRCA2 (P=0.02) at mRNA expression level. However, FA–BRCA pathway functionality demonstrated no correlation to MMC sensitivity. In cell lines, weak intensity staining of BLM by IHC correlated to high sensitivity (P=0.04) to MMC. Low BLM protein expression was significantly associated with an improved survival in patients after CRS and HIPEC (P=0.04).

Conclusions:

Low BLM levels are associated with high MMC sensitivity and an improved survival after HIPEC.     

BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years

Background:

Triple-negative (TN) tumours are the predominant breast cancer subtype in BRCA1 mutation carriers. Recently, it was proposed that all individuals below 50 years of age with TN breast cancer should be offered BRCA testing. We have evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer.

Methods:

We undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history.

Results:

BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England.

Conclusion:

Women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services.     

Triple-negative breast cancer: BRCAness and concordance of clinical features with BRCA1-mutation carriers

Background:

BRCAness is defined as shared tumour characteristics between sporadic and BRCA-mutated cancers. However, how to exactly measure BRCAness and its frequency in breast cancer is not known. Assays to establish BRCAness would be extremely valuable for the clinical management of these tumours. We assessed BRCAness characteristics frequencies in a large cohort of triple-negative breast cancers (TNBCs).

Methods:

As a measure of BRCAness, we determined a specific BRCA1-like pattern by array Comparative Genomic Hybridisation (aCGH), and BRCA1 promoter methylation in 377 TNBCs, obtained from 3 different patient cohorts. Clinicopathological data were available for all tumours, BRCA1-germline mutation status and chemotherapy response data were available for a subset.

Results:

Of the tumours, 66–69% had a BRCA1-like aCGH profile and 27–37% showed BRCA1 promoter methylation. BRCA1-germline mutations and BRCA1 promoter methylation were mutually exclusive events (P=1 × 10⁻⁵). BRCAness was associated with younger age and grade 3 tumours. Chemotherapy response was significantly higher in BRCA1-mutated tumours, but not in tumours with BRCAness (63% (12 out of 19) vs 35% (18 out of 52) pathological complete remission rate, respectively).

Conclusion:

The majority of the TNBCs show BRCAness, and those tumours share clinicopathological characteristics with BRCA1-mutated tumours. A better characterisation of TNBC and the presence of BRCAness could have consequences for both hereditary breast cancer screening and the treatment of these tumours.     

BRCA1 tumours correlate with a HIF-1�� phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression

Background:

There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers.

Methods:

Immunoperoxidase staining for HIF-1α, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes.

Results:

BRCA1 tumours correlated with positivity for HIF-1α (P=0.008) and negativity for PHD3 (P=0.037). HIF-1α positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1α expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1α or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively).

Conclusions:

The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1α or its downstream targets.     

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer

Background:

Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH).

Methods:

Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines.

Results:

Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10⁻¹¹). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10⁻⁵ and 10⁻²⁹), and identified breast and pancreatic cell lines with BRCA defects.

Conclusion:

The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.     

BRCA1 expression and improved survival in ovarian cancer patients treated with intraperitoneal cisplatin and paclitaxel: a Gynecologic Oncology Group Study

Background:

Breast cancer 1, early onset (BRCA1) is a tumour-suppressor gene associated with familial epithelial ovarian cancer (EOC). Reduced BRCA1 expression is associated with enhanced sensitivity to platinum-based chemotherapy. We sought to examine the prognostic relevance of BRCA1 expression in EOC patients treated with intraperitoneal platinum/taxane.

Methods:

The GOG-172 was a phase III, multi-institutional randomised trial of intravenous paclitaxel and cisplatin (IV therapy) vs intravenous paclitaxel, intraperitoneal cisplatin plus paclitaxel (IP therapy) in patients with optimally resected stage III EOC. The BRCA1 expression was assessed with immunohistochemistry (IHC) staining blinded to clinical outcome in archival tumour specimens. Slides with ⩽10% staining were defined as aberrant and >10% as normal. Correlations between BRCA1 expression and progression-free survival (PFS) and overall survival (OS) were analysed using Kaplan–Meier method and Cox regression analysis.

Results:

Of the 393 patients, 189 tumours had aberrant expression, and 204 had normal BRCA1 expression. There was an interaction between BRCA1 expression and route of administration on OS (P=0.014) but not PFS (P=0.054). In tumours with normal BRCA1 expression, the median OS was 58 months for IP group vs 50 months for IV group (P=0.818). In tumours with aberrant BRCA1 expression, the median OS was 84 vs 47 months in the IP vs IV group, respectively (P=0.0002). Aberrant BRCA1 expression was an independent prognostic factor for better survival in women randomised to IP therapy (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.47–0.97, P=0.032). Similar survival was observed in the IV and IP patients with normal BRCA1 expression. Multivariate but not univariate modelling demonstrated that IV patients with aberrant vs normal BRCA1 expression had worse survival.

Conclusion:

Decreased BRCA1 expression is associated with a 36-month survival improvement in patients with EOC treated with IP chemotherapy. Although these results merit validation in future studies, the results suggest that decreased BRCA1 expression predicts for improved response to cisplatin-based IP chemotherapy with cisplatin and paclitaxel.