Alaa Alshareeda

Clinicopathological significance of KU70/KU80, a key DNA damage repair protein in breast cancer.

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.

SUMOylation proteins in breast 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.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast 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<0.0001), but this effect was not independent of other prognostic variables.Conclusions:This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Checkpoint kinase1 (CHK1) is an important biomarker in breast cancer having a role in chemotherapy response

Background:Checkpoint kinase1 (CHK1), which is a key component of DNA-damage-activated checkpoint signalling response, may have a role in breast cancer (BC) pathogenesis and influence response to chemotherapy. This study investigated the clinicopathological significance of phosphorylated CHK1 (pCHK1) protein in BC.Method:pCHK1 protein expression was assessed using immunohistochemistry in a large, well-characterized annotated series of early-stage primary operable invasive BC prepared as tissue microarray (n=1200).Result:pCHK1 showed nuclear and/or cytoplasmic expression. Tumours with nuclear expression showed positive associations with favourable prognostic features such as lower grade, lower mitotic activity, expression of hormone receptor and lack of expression of KI67 and PI3K (P<0.001). On the other hand, cytoplasmic expression was associated with features of poor prognosis such as higher grade, triple-negative phenotype and expression of KI67, p53, AKT and PI3K. pCHK1 expression showed an association with DNA damage response (ATM, RAD51, BRCA1, KU70/KU80, DNA-PKCα and BARD1) and sumoylation (UBC9 and PIASγ) biomarkers. Subcellular localisation of pCHK1 was associated with the expression of the nuclear transport protein KPNA2. Positive nuclear expression predicted better survival outcome in patients who did not receive chemotherapy in the whole series and in ER-positive tumours. In ER-negative and triple-negative subgroups, nuclear pCHK1 predicted shorter survival in patients who received cyclophosphamide, methotrexate and 5-florouracil chemotherapy.Conclusions:Our data suggest that pCHK1 may have prognostic and predictive significance in BC. Subcellular localisation of pCHK1 protein is related to its function.

Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer

Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor–negative (ER−), progesterone receptor–negative (PR−), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER+/Her2−/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER+/Her2−/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer–specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer. Mol Cancer Ther; 14(4); 1057–65. ©2015 AACR.

Abstract B120: Ada3, a component of ATAC complex is involved in regulation of the Genomic stability, DNA repair process and breast cancer

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity which further leads to oncogenesis. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, as loss of Ada3 results in mouse embryonic lethality. Furthermore cre-mediated deletion of Ada3 in Ada3fl/ fl mouse embryonic fibroblasts (MEFs), led to enhanced chromosomal aberrations, which further increased upon DNA damage Significantly, Ada3 loss was associated with increased levels of phospho-ATM ), γH2AX, 53BP1 andRAD51 although radiation response was intact in Ada3-/- cells. Notably, Ada3-/- cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Given the frequent dysregulation of cell cycle regulatory components in human cancer, we used immunohistochemistry of paraffin embedded tissues to examine Ada3 expression in breast cancer tissue. These analyses showed predominant nuclear Ada3 staining correlated with ER+ status,whereas predominant cytoplasmic Ada3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic Ada3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear Ada3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of Ada3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. These results show nuclearAda3 staining in breast cancer tissues correlates with ER+ expression and together serve as a marker of good prognosis, whereas predominant cytoplasmic Ada3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Our current efforts are focused on dissecting out the molecular pathways for cytoplasmic Ada3. The hypothesis that we are testing are i) Is there a role of second isoform of Ada3 that is predominantly cytoplasmic or ii) posttranslational modification (such as phosphorylation, acetylation or ubiquitination) of Ada3 regulates cytoplasmic expression of Ada3. Citation Format: Sameer Mirza, Shakur Mohibi, Bryan J. Katafiasz, Jun Wang, Bhavana J. Dave, Emad A. Rakha, Alaa Alshareeda, Hamid Band, Vimla Band. Ada3, a component of ATAC complex is involved in regulation of the Genomic stability, DNA repair process and breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B120.

Abstract 4593: Expression of Ecdysoneless in breast cancer progression predicts poor outcome in breast cancer patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is the most common type of cancer and second leading cause of cancer-related deaths in women in the United States. Our laboratory has identified the protein hEcd (human ortholog of Drosophila Ecdysoneless) as a novel regulator of cell cycle. Previous studies showed that Ecd regulates cell cycle by regulating the Rb-E2F pathway. Given the dysregulation of the cell cycle machinery in cancer, we examined expression of Ecd in normal, benign, ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDCs) of breast. In first cohort, we showed while normal and hyperplasia of breast barely showed Ecd expression, high Ecd expression was observed in DCIS and IDC patient tissue specimens. In this cohort of 104 IDC patents, Ecd expression was positively associated with higher grade (p=0.04).We then analyzed Ecd expression in a second larger cohort (954) and observed similar results, where increased Ecd expression was associated with tumors of higher histological grade (p=0.013), mitotic count (p=0.032), and Nottingham Prognostic Index score (p=0.014). Notably, Ecd expression was positively associated with HER2/neu (p=0.002) overexpression. Significantly, a positive association between Ecd expression and shorter breast cancer specific survival (BCSS) (p=0.008) and disease-free survival (DFS) (p=0.003) was observed in HER2/neu overexpressing patients. Taken together, our results demonstrate Ecd expression as a novel marker for breast cancer progression that predicts tumor progression and the clinical outcome in breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4593. doi:1538-7445.AM2012-4593