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PDF file - 322K, Supplemental Figure 1. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancer whole cohort (A), Lymph node negative (B) and lymphnode positive (C). Supplemental Figure 2. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancerlow risk, no adjuvant therapy (A), high risk, received endocrine therapy (B) and high risk, received CMF chemotherapy (C). Supplemental Figure 3. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancerER negative tumours (A), Triple negative tumours (B). Supplemental Figure 4. Clonogenic survival assay (A), Annexin-V FITC assay (B).

PDF file - 104K, Supplemental Table S1: Clinicopathological characteristics of whole breast cancer cohort Supplemental Table S2: Clinicopathological characteristics of validation setSupplementary Table S3: Antigens, primary antibodies, clone, source, optimal dilution and scoring system used for each immunohistochemical marker Supplementary Table S4: siRNA constructs for XRCC1 knockdown

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n = 1,297) and validated in an independent cohort of estrogen receptor (ER)-α–negative breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia–mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G2–M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5); 1621–34. ©2012 AACR.

The identification of clinically-relevant early diagnostic and prognostic protein biomarkers is essential to maximize therapeutic efficacy and prevent cancer progression. The aim of the current study is to determine whether aberrant plasma protein profile can be applied as a surrogate tool for early diagnosis of bladder carcinoma. Plasma samples from patients with low grade non-muscle invasive bladder cancer and healthy controls were analyzed using combined 2D-DIGE and mass-spectrometry to identify differentially expressed proteins. Validation was performed using western blotting analysis in an independent cohort of cancer patients and controls. Fifteen differentially-expressed proteins were identified of which 12 were significantly up-regulated and three were significantly down-regulated in tumors compared to controls. The Ingenuity Pathways Analysis revealed functional connection between the differentially-expressed proteins and immunological disease, inflammatory disease and cancer mediated through chemokine and cytokine signaling pathway and NF-kB transcription factor. Among the three validated proteins, haptoglobin was able to distinguish between patients with low grade bladder cancer and the controls with high sensitivity and specificity (AUC > 0.87). In conclusion, several biomarker proteins were identified in bladder cancer. Haptoglobin is a potential candidate that merit further investigation to validate its usefulness and functional significance as potential biomarkers for early detection of bladder cancer.

Background: Bladder cancer is a life-threatening disease and a major cause of cancer-associated complications. The main challenges confronted during the clinical management of bladder cancer are associated with recurrence and disease progression to the muscle-invasive phenotype. Improved early detection of the disease is of paramount importance to prevent disease progression and improve survival. Hence, novel clinically applicable biomarkers for early detection are warranted. Methods: In the current study, a comparative proteomic approach was undertaken using plasma samples to identify protein biomarkers associated with the muscle-invasive phenotype of bladder carcinoma. Isolated plasma proteins were depleted, DIGE-labeled, then subjected to conventional 2D electrophoresis followed by mass spectrometry for identification of differentially expressed proteins. Western blot was used for data validation. Results: Fourteen differentially expressed proteins with statistically significant changes in abundance between the cancer group and control group were identified. Three differentially expressed proteins were selected for validation, among which apolipoprotein A1 exhibited high specificity and sensitivity (AUC = 0.906). Ingenuity pathway analysis identified IFN-γ and TNF-α as the main signaling hub for the differentially regulated proteins. Conclusion: Our findings provide additional insight into understanding bladder cancer pathogenesis. Our data identified potential non-invasive plasma-derived biomarker proteins that merit additional investigation to validate its clinical usefulness to prevent bladder cancer progression.

Background The glutathione S-transferases (GSTs) are a superfamily of phase II detoxifying enzymes that inactivates a wide variety of potential carcinogens through glutathione conjugation. Polymorphic changes in the GST genes have been reported to be associated with increased susceptibility to cancer development and anticancer drug resistance. In this study, we investigated the association between genetic variants in GSTM1 and GSTP1 and patients’ clinicopathological parameters. The prognostic values of such associations were evaluated among bladder cancer patients. Methods Genotyping of GSTM1 and GSTP1 in bladder cancer patients was assessed using polymerase chain reaction followed by DNA sequencing. Overall survival was estimated using the Kaplan-Meier method and multiple logistic regression and correlation analysis were performed. Results The GSTM1 null genotype was significantly associated with poor overall survival compared with the wild-type GSTM1 genotype. There was a trend towards better overall survival in patients with wild-type GSTP1 allele (AA) compared with GSTP1 (AG/GG) genotype. Interestingly, Kaplan-meier survival curve for GSTM1 null patients adjusted for sub-cohort with amplified HER2 gene showed poor survival compared with the GSTM1 null/ non-amplified HER2 gene. Also the same population when adjusted with HER2 protein expression, data showed poor survival for patients harboring GSTM1 null/high HER2 protein expression compared with low protein expression. Conclusion This study focuses on the impact of GSTM1 null genotype on bladder cancer patients’ outcome. Further investigations are required to delineate the underlying mechanisms of combined GSTM−/− and HER2 status in bladder cancer.

Background: It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. Methods: A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. Results: KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P

BRCA1, a key factor in homologous recombination (HR) repair may also regulate base excision repair (BER). Targeting BRCA1?BER deficient cells by blockade of ATM and DNA?PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol ? protein expression in two cohorts (n = 1602 sporadic and n = 50 germ?line BRCA1 mutated) and mRNA expression in two cohorts (n = 1952 and n = 249). Artificial neural network analysis for BRCA1?DNA repair interacting genes was conducted in 249 tumours. Pre?clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA?PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol ? at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol ? expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol ? expressing BRCA1 negative tumours (ps < 0.05). Pre?clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol ?. BRCA1?BER deficient cells were sensitive to ATM and DNA?PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol ? expression status in BRCA1 negative tumours may have prognostic significance. BRCA1?BER deficient cells could be targeted by ATM or DNA?PKcs inhibitors for personalized therapy.

Although the role of BRCA1 and the homologous recombination (HR) pathway in breast cancer (BC) has been extensively studied, the alternative repair pathway for DNA double-strand breaks (DSBs), non-homologous end-joining (NHEJ) remains to be defined. Ku proteins bind to DNA DSB ends and play a key role in NHEJ. In this study we aimed to assess the expression and biological significance of the KU70/KU80 heterodimer in the different molecular classes of BC. The expression of KU70/KU80 was assessed immunohistochemically in a well-characterised and annotated series of 1302 unselected invasive BC cases with a long-term follow-up together with 25 cases with known BRCA1 mutations. The results were correlated with clinicopathological parameters, other DNA repair proteins and patient outcome. The expression of KU70/KU80 protein was further evaluated in various BC cell lines using western blotting and reverse-phase protein microarray (RPPA). Nuclear KU70/KU80 expression was correlated with features of poor prognosis including higher histological grade, lymphovascular invasion, negative oestrogen receptor expression, basal-like phenotype, P53 and CHK1 positivity. KU70/KU80 was expressed in all BRCA1-associated tumours and showed an inverse correlation with nuclear BRCA1 protein and aberrant cytoplasmic RAD51 expression. RPPA confirmed these results and showed higher expression of KU70/KU80 in BRCA1-deficient cell line compared to BRCA1-proficient cell line. KU70/KU80 expression showed an association with disease-free interval; however, it was not an independent predictor of outcome. As a conclusion, KU70/KU80 may play a role in DNA DSBs repair in HR-deficient tumours. Further study of other NHEJ markers in sporadic BC is warranted.

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n = 1,297) and validated in an independent cohort of estrogen receptor (ER)-α–negative breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia–mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G2–M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5); 1621–34. ©2012 AACR.

FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p = 4.89 × 10(-57)), high mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)), PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)), integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5 (p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p = 9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps < 0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps < 0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps < 0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps < 0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer.

Small Ubiquitin-like Modifier proteins (or SUMO) modify the function of protein substrates involved in various cellular processes including DNA damage response (DDR). It is becoming apparent that dysregulated SUMO contribute to carcinogenesis by affecting post-transcriptional modification of key proteins. It is hypothesised that SUMO contributes to the aggressive nature of breast cancer particularly those associated with features similar to breast carcinoma arising in patients with BRCA1 germline mutations. This study aims to assess the clinical and biological significance of three members of SUMO in a well-characterised annotated series of BC with emphasis on DDR. The study cohort comprised primary operable invasive BC including tumours from patients with known BRCA1 germline mutations. SUMO proteins PIAS1, PIAS4 and UBC9 were assessed using immunohistochemistry utilising tissue microarray technology. Additionally, their expression was assessed using reverse phase protein microarray utilising different cell lines. PIAS1 and UBC9 showed cytoplasmic and/or nuclear expression while PIAS4 was detected only in the nuclei. There was a correlation between subcellular localisation and expression of the nuclear transport protein KPNA2. Tumours showing positive nuclear/negative cytoplasmic expression of SUMO featured good prognostic characteristics including lower histologic grade and had a good outcome. Strong correlation with DDR-related proteins including BRCA1, Rad51, ATM, CHK1, DNA-PK and KU70/KU80 was observed. Correlation with ER and BRCA1 was confirmed using RPPA on cell lines. SUMO proteins seem to play important role in BC. Not only expression but also subcellular location is associated with BC phenotype.

Short arm of chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications in breast cancer. Although such genetic changes may have phenotypic consequences, the identity of candidate gene(s) remains to be clearly defined. Pol β gene is localized to chromosome 8p12‐p11 and encodes a key DNA base excision repair protein. Pol β may be a tumour suppressor and involved in breast cancer pathogenesis. We conducted the first and the largest study to comprehensively evaluate pol β in breast cancer. We investigated pol β gene copy number changes in two cohorts (n = 128 & n = 1952), pol β mRNA expression in two cohorts (n = 249 & n = 1952) and pol β protein expression in two cohorts (n = 1406 & n = 252). Artificial neural network analysis for pol β interacting genes was performed in 249 tumours. For mechanistic insights, pol β gene copy number changes, mRNA and protein levels were investigated together in 128 tumours and validated in 1952 tumours. Low pol β mRNA expression as well as low pol β protein expression was associated high grade, lymph node positivity, pleomorphism, triple negative, basal‐like phenotypes and poor survival (ps

Uracil in DNA is an important cause of mutagenesis. SMUG1 is a uracil-DNA glycosylase that removes uracil through base excision repair. SMUG1 also processes radiation-induced oxidative base damage as well as 5-fluorouracil incorporated into DNA during chemotherapy. We investigated SMUG1 mRNA expression in 249 primary breast cancers. SMUG1 protein expression was investigated in 1,165 breast tumours randomised into two cohorts [training set (n = 583) and test set (n = 582)]. SMUG1 and chemotherapy response was also investigated in a series of 315 ER-negative tumours (n = 315). For mechanistic insights, SMUG1 was correlated to biomarkers of aggressive phenotype, DNA repair, cell cycle and apoptosis. Low SMUG1 mRNA expression was associated with adverse disease specific survival (p = 0.008) and disease-free survival (p = 0.008). Low SMUG1 protein expression (25 %) was associated with high histological grade (p < 0.0001), high mitotic index (p < 0.0001), pleomorphism (p < 0.0001), glandular de-differentiation (p = 0.0001), absence of hormonal receptors (ER−/PgR−/AR) (p < 0.0001), presence of basal-like (p < 0.0001) and triple-negative phenotypes (p < 0.0001). Low SMUG1 protein expression was associated with loss of BRCA1 (p < 0.0001), ATM (p < 0.0001) and XRCC1 (p < 0.0001). Low p27 (p < 0.0001), low p21 (p = 0.023), mutant p53 (p = 0.037), low MDM2 (p < 0.0001), low MDM4 (p = 0.004), low Bcl-2 (p = 0.001), low Bax (p = 0.003) and high MIB1 (p < 0.0001) were likely in low SMUG1 tumours. Low SMUG1 protein expression was associated with poor prognosis in univariate (p < 0.001) and multivariate analysis (p < 0.01). In ER+ cohort that received adjuvant endocrine therapy, low SMUG1 protein expression remains associated with poor survival (p < 0.01). In ER− cohort that received adjuvant chemotherapy, low SMUG1 protein expression is associated with improved survival (p = 0.043). Our study suggests that low SMUG1 expression may correlate to adverse clinicopathological features and predict response to adjuvant therapy in breast cancer.