Differential expression and regulation of ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12 gene profiles in colon cancer tissues and their in vitro response to epigenetic drugs.

Colon cancer (CC) is a significant cause of death worldwide, particularly in Saudi Arabia. To increase the accuracy of diagnosis and treatment, it is important to discover new specific biomarkers for CC. The main objectives of this research are to identify potential specific biomarkers for the early diagnosis of CC by analyzing the expressions of eight cancer testis (CT) genes, as well as to analyze how epigenetic mechanisms control the expression of these genes in CC cell lines. Tissue samples were collected from 15 male patients with CC tissues and matched NC tissues for gene expression analysis. The expression levels of specific CT genes, including ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12, were assessed using quantitative techniques. To validate the gene expression patterns, we used publicly available CC statistics. To investigate the effect of inhibition of DNA methylation and histone deacetylation on CT gene expression, in vitro experiments were performed using HCT116 and Caco-2 cell lines. There was no detected expression of the genes neither in the patient samples nor in NC tissues, except for TEX48, which exhibited upregulation in CC samples compared to NC tissues in online datasets. Notably, CT genes showed expression in testis samples. In vitro, experiments demonstrated significant enhancement in mRNA expression levels of ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12 following treatment with 5-aza-2'-deoxycytidine and trichostatin A in HCT116 and Caco-2 cell lines. Epigenetic treatments modify the expression of CT genes, indicating that these genes can potentially be used as biomarkers for CC. The importance of conducting further research to understand and target epigenetic mechanisms to improve CC treatment cannot be overemphasized.

Hidden regulators: the emerging roles of lncRNAs in brain development and disease.

Long non-coding RNAs (lncRNAs) have emerged as critical players in brain development and disease. These non-coding transcripts, which once considered as "transcriptional junk," are now known for their regulatory roles in gene expression. In brain development, lncRNAs participate in many processes, including neurogenesis, neuronal differentiation, and synaptogenesis. They employ their effect through a wide variety of transcriptional and post-transcriptional regulatory mechanisms through interactions with chromatin modifiers, transcription factors, and other regulatory molecules. Dysregulation of lncRNAs has been associated with certain brain diseases, including Alzheimer's disease, Parkinson's disease, cancer, and neurodevelopmental disorders. Altered expression and function of specific lncRNAs have been implicated with disrupted neuronal connectivity, impaired synaptic plasticity, and aberrant gene expression pattern, highlighting the functional importance of this subclass of brain-enriched RNAs. Moreover, lncRNAs have been identified as potential biomarkers and therapeutic targets for neurological diseases. Here, we give a comprehensive review of the existing knowledge of lncRNAs. Our aim is to provide a better understanding of the diversity of lncRNA structure and functions in brain development and disease. This holds promise for unravelling the complexity of neurodevelopmental and neurodegenerative disorders, paving the way for the development of novel biomarkers and therapeutic targets for improved diagnosis and treatment.

FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are highly expressed in colon cancer patients and are regulated in vitro by epigenetic alterations.

Background: Colon cancer is a serious public health issue and a major cause of cancer-related mortality worldwide, including Saudi Arabia. Knowledge of genes associated with colon cancer development and progression is essential for identifying new cancer-specific biomarkers to improve the diagnosis of colon cancer. Methods: The expression levels of FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 in 15 adjacent colon cancer and normal colon tissue samples from male patients were investigated using reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) assays. qRT-PCR analysis was also used to determine whether reducing DNA methyltransferase (via 5-aza-2'-deoxycytidine treatment) or histone deacetylation (via trichostatin treatment) increased the expression levels of the tested genes. Results: The analysis of the 15 colon cancer and adjacent normal colon tissue samples revealed that all six genes were expressed in both groups, but their expression levels were significantly higher in the colon cancer group. Furthermore, the mRNA expression levels of the FTHL17, PRM2, CABYR, CPXCR1, and ADAM29 genes were considerably upregulated after treatment of HCT116 and Caco-2 cells with 5-aza-2'-deoxycytidine and trichostatin. However, the CABS1 gene was activated only with trichostatin treatment. Conclusions: The findings of this study suggest that FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are suitable candidate biomarkers of colon cancer and their expressions are regulated by hypomethylation and hyperacetylation.

Homologous recombination mRNAs (RAD21, RAD50 and BARD1) have a potentially poor prognostic role in ERBB2-low bladder cancer patients.

Human epidermal growth factor receptor 2 (HER2/ERBB2) factor is known to be implicated in many malignancies and the potential of it as a prognostic biomarker was reported years ago. Molecular subtypes of HER2/ERBB2 negative and positive with distinct clinical outcomes have been identified in recent years; however, it is still under investigation for bladder cancer. This study evaluates the biological and prognostic significance of RAD21, RAD50 and BARD1 (homologous recombination biomarkers) mRNA levels with ERBB2 low and high expression to explore their impact on bladder cancer patient survival and cancer aggressiveness. The expression of ERBB2, RAD21, RAD50 and BARD1 mRNA levels was assessed in The Cancer Genome Atlas (TCGA) bladder cancer dataset along with four validation cohorts. Outcome analysis was evaluated using disease-free survival (DFS) and overall survival (OS). Univariate and multivariate analysis were used to evaluate the relationship between RAD21, RAD50, BARD1 and ERBB2 expression and clinicopathological variables. A significant increase in mRNA expression levels of RAD21, RAD50 and BARD1 was noticed in ERBB2-low patients compared to ERBB2-high patients. This overexpression of the homologous recombination repair transcripts was associated with poor outcome in ERBB2-low tumors, not in ERBB2-high tumors. Furthermore, the combined expression of high RAD21/RAD50, high RAD21/BARD1 or high RAD50/BARD1 were significantly associated with worse DFS and a better outcome for those with low co-expression in the ERBB2-low cohort. High expression of either RAD21/RAD50 or RAD21/BARD1 in ERBB2-low cohort associated with higher chance of metastasis. In addition, gene expression of BARD1 alone or in combination with RAD50 acted as an independent prognostic factor for worst survival. The data presented in this study reveal a connection between RAD21, RAD50, BARD1 and ERBB2 and patient survival. Importantly, it provided novel findings and potential prognostic markers, particularly in ERBB2-low bladder cancer.

Nanotechnology: A revolutionary approach to prevent breast cancer recurrence.

Breast cancer is the most prevalent malignancy in women worldwide and one of the deadliest after lung cancer. Currently, standard treatment approaches for breast cancers are surgery accompanied by chemotherapy or radiotherapy. Cancer local recurrence after mastectomy is commonly considered as being a poor prognostic predictor. There have been advancements in the procedures utilized for breast reconstruction following mastectomy, much as there have been advancements in the early diagnosis and treatment of breast cancer. For the last decade, developing nanotechnology applications for cancer therapies has had much focus. The benefits granted by nanotechnologies via enhancing biological processes and promoting better biomaterial compatibility, as well as generating functionalized tissues, transpire exciting possibilities. Modified nanomedicine may introduce tremendous improvements to the fields of breast cancer recurrence through implants. It can modify the surfaces of implants to optimize tissue growth, thus minimizing inflammation and unsatisfactory results. Here we discuss new nanotechnology advancements and incorporate them into breast reconstruction surgeries following mastectomy or lumpectomy. In addtion, we repurpose old technologies, like growth factor therapies using nanotechnology for more efficient delivery.

Preparing the Bone Tissue Regeneration Ground by Exosomes: From Diagnosis to Therapy.

Bone tissue engineering employs acellular scaffolds or scaffolds, along with cells and growth factors, to provide the mechanical support needed, as well as serve as a delivery vehicle for bioactive molecules to the injury sites. As tissue engineering continues to evolve, it has integrated two emerging fields: stem cells and nanotechnology. A paracrine factor that is found to be responsible for the major regenerative effect in stem cell transplantation is an extracellular vesicle called an 'exosome'. Recent advances in nanotechnology have allowed the 'exosome' to be distinguished from other extracellular vesicles and be polymerized into a well-defined concept. Scientists are now investigating exosome uses in clinical applications. For bone-related diseases, exosomes are being explored as biomarkers for different bone pathologies. They are also being explored as a therapeutic agent where progenitor cell-derived exosomes are used to regenerate damaged bone tissue. In addition, exosomes are being tested as immune modulators for bone tissue inflammation, and finally as a delivery vehicle for therapeutic agents. This review discusses recently published literature on the clinical utilization of exosomes in bone-related applications and the correlated advantages. A particular focus will be placed on the potential utilization of regenerative cell-derived exosomes as a natural biomaterial for tissue regeneration.

Cancer of Unknown Primary Site: Real Entity or Misdiagnosed Disease?

Metastasis is a late event in the progression of any tumour. However, invasive cancers are occasionally detected in the form of metastatic lesions without a clearly detectable primary tumour. Cancer of unknown primary site (CUP) is defined as a confirmed metastatic tumour, with unknown primary tumour site, despite the standardized diagnostic approach that includes clinical history, routine laboratory tests, and complete physical examination. Due to the lack of basic research on its primary causes, CUP is appropriately termed an 'orphan' cancer. Nevertheless, CUP accounts for 2-5% of diagnosed malignancies. To date, it is unclear whether CUP is an entity with primary dormancy as its hallmark or an entity with genetic abnormalities that cause it to manifest as a primary metastatic disease. In this review, we discuss different aspects of CUP, including its current diagnostic methods, angiogenesis effectors, relationship with cancer stem cells and current treatments.

Scaffold-Free 3-D Cell Sheet Technique Bridges the Gap between 2-D Cell Culture and Animal Models.

Various tissue engineering techniques have been created in research spanning two centuries, resulting in new opportunities for growing cells in culture and the creation of 3-D tissue-like constructs. These techniques are classified as scaffold-based and scaffold-free techniques. Cell sheet, as a scaffold-free technique, has attracted research interest in the context of drug discovery and tissue repair, because it provides more predictive data for in vivo testing. It is one of the most promising techniques and has the potential to treat degenerative tissues such as heart, kidneys, and liver. In this paper, we argue the advantages of cell sheets as a scaffold-free approach, compared to other techniques, including scaffold-based and scaffold-free techniques such as the classic systemic injection of cell suspension.

The effect of human placental chorionic villi derived mesenchymal stem cell on triple-negative breast cancer hallmarks.

Mesenchymal stem cells (MSCs) can influence the tumour microenvironment (TEM) and play a major role in tumourigenesis. Triple-negative [Ostrogen receptor (ER-), Progesterone receptor (PgR-), and HER2/neu receptor (HER2-)] breast cancer (TNBC) is an aggressive class of BC characterized by poor prognosis and lacks the benefit of routinely available targeted therapies. This study aims to investigate the effect of human placental chorionic villi derived MSCs (CVMSCs) on the behavior of TNBC in vitro. This was done by assaying different cancer hallmarks including proliferation, migration and angiogenesis. Cell proliferation rate of TNBC cell line (MDA-MB231) was monitored in real time using the xCELLigence system. Whereas, Boyden chamber migration assay was used to measure MDA-MB231 motility and invasiveness toward CVMSCs. Finally, a three-dimensional (3D) model using a co-culture system of CVMSCs with MDA-MB231 with or without the addition of human umbilical vein endothelial cells (HUVECs) was created to assess tumour angiogenesis in vitro. CVMSCs were able to significantly reduce the proliferative and migratory capacity of MDA-MB231 cells. Co-culturing of MDA-MB231 with CVMSCs, not only inhibited the tube formation ability of HUVECs but also reduced the expression of the BC characteristic cytokines; IL-10, IL-12, CXCL9 and CXCL10 of CVMSCs. These results support the hypothesis that CVMSCs can influence the behavior of TNBC cells and provides a basic for a potential therapeutic approach in a pre-clinical settings. The data from this study also highlight the complexity of the in vitro cancer angiogenesis model settings and regulations.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

The potential of cell sheet technique on the development of hepatocellular carcinoma in rat models.

BACKGROUND: Hepatocellular carcinoma (HCC) is considered the 3rd leading cause of death by cancer worldwide with the majority of patients were diagnosed in the late stages. Currently, there is no effective therapy. The selection of an animal model that mimics human cancer is essential for the identification of prognostic/predictive markers, candidate genes underlying cancer induction and the examination of factors that may influence the response of cancers to therapeutic agents and regimens. In this study, we developed a HCC nude rat models using cell sheet and examined the effect of human stromal cells (SCs) on the development of the HCC model and on different liver parameters such as albumin and urea. METHODS: Transplanted cell sheet for HCC rat models was fabricated using thermo-responsive culture dishes. The effect of human umbilical cord mesenchymal stromal cells (UC-MSCs) and human bone marrow mesenchymal stromal cells (BM-MSCs) on the developed tumour was tested. Furthermore, development of tumour and detection of the liver parameter was studied. Additionally, angiogenesis assay was performed using Matrigel. RESULTS: HepG2 cells requires five days to form a complete cell sheet while HepG2 co-cultured with UC-MSCs or BM-MSCs took only three days. The tumour developed within 4 weeks after transplantation of the HCC sheet on the liver of nude rats. Both UC-MSCs and BM-MSCs improved the secretion of liver parameters by increasing the secretion of albumin and urea. Comparatively, the UC-MSCs were more effective than BM-MSCs, but unlike BM-MSCs, UC-MSCs prevented liver tumour formation and the tube formation of HCC. CONCLUSIONS: Since this is a novel study to induce liver tumour in rats using hepatocellular carcinoma sheet and stromal cells, the data obtained suggest that cell sheet is a fast and easy technique to develop HCC models as well as UC-MSCs have therapeutic potential for liver diseases. Additionally, the data procured indicates that stromal cells enhanced the fabrication of HepG2 cell sheets. This provides the foundation for future research using stromal cells in preclinical and clinical investigations.

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein.

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51 N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N(+) was an independent predictor of longer BCSS (P < 0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients' outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC.

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.

Biological and clinical significance of PARP1 protein expression in breast cancer.

Poly(ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair. PARP inhibitors have gained recent attention as promising therapeutic agents for the treatment of solid tumours including breast cancer (BC). However, the biological and clinical significance of PARP1 expression in BC and its role in DNA-damage response (DDR) remain to be defined. We investigated the expression of PARP1 expression, cleaved (PARP1c) and non-cleaved (PAR1nc) forms, in a large and well-characterised cohort of clinically annotated stage I-III operable BCs (n = 1,269) and 43 BRCA1-mutated BCs using immunohistochemistry. PARP1 expression was correlated to clinicopathological variables, outcome and expression of other key DNA repair proteins (BRCA1, RAD51, Ku70/80, PIASγ and CHK1). Expression of PARP1 was exclusively nuclear. 49 and 85 % of sporadic BC showed expression PARP1nc and PARP1c, respectively. In BRCA1-mutated tumours, PARP1nc/PARP1c was highly expressed (95 and 79 %, respectively). PARP1nc expression was positively associated with premenopausal younger age patients, larger size and higher tumour grade. PARP1 was positively associated with DDR-proteins; RAD51, BRCA1, CHK1 and PIASγ (p < 0.001). Negative association was found between PARP1nc and Ki67. PARP1c was associated with ER (p < 0.001). Different associations between PARP1 and DDR-proteins were observed when stratified based on ER/BRCA1 status. PARP1 was not an independent predictor of outcome in sporadic or BRCA1-mutated BC. Our results demonstrate a potential biological role for PARP1c and PARP1nc in DNA repair in BC based on the significant association with other key DNA damage repair proteins. These associations were not restricted to ER-negative or triple-negative subgroup.

Prognostic and biological significance of proliferation and HER2 expression in the luminal class of breast cancer.

The definition of Luminal-B subclass of breast cancer (BC) varies in literature. In this study, we have compared the proliferation status; assessed using KI67 labeling index (KI67-LI), and HER2-expression in estrogen receptor positive (ER+) BC to assess their impact on the biological and clinical characteristics of luminal-BC. 1547 (73.8 %) well-characterized clinically annotated stage I-III ER + BC were assessed for expression of KI67, HER2 (ASCO guidelines), and a large panel of relevant biomarkers (no = 37). 46.3 % of the cases show high KI67-LI (>13 %) and 8.4 % show HER2+ and both markers are positively associated with younger age, higher tumor grade and poorer outcome. High KI67-LI and HER2+ are associated with upregulation of ER-coactivators and proliferation-related markers and with downregulation of good prognostic markers. High KI67-LI is associated with larger size, advanced stage, and lymphovascular invasion (LVI) and with downregulation of luminal-enriched and DNA-damage repair markers. In contrast, HER2+ is associated with upregulation of ER-regulated proteins and E-cadherin. When analysis is restricted to high KI67-LI subgroup, HER2+ shows an association with upregulation of differentiation-associated proteins and E-cadherin. Conversely, within HER2+ class, high KI67-LI maintains its association with downregulation of differentiation-associated/luminal-enriched proteins. Outcome analyses indicate that both markers are independently associated with shorter survival but HER2+ is associated with a worse outcome. Although both are associated with high proliferation and poor prognosis within ER + BC, HER2+ is less frequent than high KI67-LI. Unlike KI67, HER2 seems to independently drive the aggressive behavior of ER+ tumors without downregulation of luminal proteins.

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.

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.

Characteristics of basal cytokeratin expression in breast cancer.

Breast cancer is recognised to be a heterogeneous disease and the second most common cause of morbidity and mortality worldwide in women. Basal-like breast cancer (BLBC) is associated with aggressive characteristics including development of recurrent disease and reduced survival. BLBC has been defined in some studies as tumours lacking both oestrogen receptor and progesterone receptor protein expression. Gene expression studies have shown that these tumours are also associated with expression of basal-type cytokeratins, the phenotypic patterns of basal cytokeratin expression in BLBC have not been widely studied. A well-characterised series of 995 invasive breast cancers with a long-term follow up were investigated using immunohistochemical staining for four basal cytokeratins (CK5, CK5/6, CK14 and CK17). The data were analysed using univariate and clustering analysis. As a result BLBC, as defined by negativity for ER and HER2 showed variable positivity for basal cytokeratin expression: 61.7 % CK5, 50.5 % CK5/6, 24.2 % CK14 and 23 % CK17. These characteristics were associated with poor outcome characteristics including high histological grade, mitosis, pleomorphism and tumour size >1.5 cm. CK5 positivity was more associated with ER(-), PgR(-), TN and double ER(-)PgR(-), than the other cytokeratins. Four different clusters of basal cytokeratin expression patterns were identified: (1) negativity for all basal cytokeratins, (2) CK5(+)/CK17(-), (3) CK5(-)/CK17(+) and (4) CK5(+)/CK17(+). These patterns of basal cytokeratin expression associated with differences in patient outcome, clusters 1 and 3 showed better outcomes than cluster 4 and 2, with cluster 2 having the poorest prognosis. In conclusion, four basal cytokeratin expression patterns were identified in human breast cancer using unsupervised clustering analysis and these patterns are associated with differences in patient outcome.

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients' outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While 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. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.

Fatty acid binding protein 7 expression and its sub-cellular localization in breast cancer.

FABP7 is a member of the multi-gene fatty acid binding protein family. It is expressed in the mammary gland and has been shown to function as inhibitor of proliferation of breast tumour cells and to promote differentiation through the JAK/Stat pathway. Cytoplasmic FABP7 expression has been shown to be associated with a favourable prognosis of basal-like breast cancer. In other tissues, varying sub-cellular localization of FABP7 between the nucleus and cytoplasm has been observed. Tissue microarray preparations of well-characterized series of 1,249 unselected and 245 ER-negative invasive breast cancers with a long-term follow-up were investigated in this study to assess the biological and clinical significance of FABP7 sub-cellular localization using immunohistochemistry. Both nuclear and cytoplasmic FABP7 were observed. Nuclear FABP7 was associated with high histologic grade, mitotic frequency, pleomorphism and stage, in addition to basal phenotype (BP) and triple-negative (TN) phenotype. Nuclear FABP7 expression showed an association with expression of markers associated with proliferation and cell-cycle control including Ki67, p53 and p21; however, cytoplasmic FABP7 was associated only with Ki67 and P53 (P = 0.001, < 0.001 respectively). Interestingly, in multivariate analysis, nuclear FABP7 expression in BP was significantly associated with longer DFI (P = 0.025) independent of cytoplasmic expression. Tumours with only nuclear positive FABP7 expression had significantly better prognosis than those with only cytoplasmic expression. This is the first study elucidating the sub-cellular localization of FABP7 in a large series of breast cancer cases. Our observations demonstrate the considerable heterogeneity in expression patterns of FABP7 within breast cancer that relates to differences in biological behaviour especially in basal-like breast cancer. Further investigation of the biology of FABP7 in breast cancer is warranted.