Clinico-Pathological and Prognostic Significance of a Combination of Tumor Biomarkers in Iranian Patients With Breast Cancer.

PURPOSE: Breast cancer is one of the most common cancers in the world. It is a multifaceted malignancy with different histopathological and biological features. Molecular biomarkers play an essential role in accurate diagnosis, classification, prognosis, prediction of treatment response, and cancer surveillance. This study investigated the clinico-pathological and prognostic significance of HER3 and ROR1 in breast cancer samples. METHODS: Tissue microarrays (TMA) were constructed using tissue blocks of 444 Iranian breast cancer patients diagnosed with breast cancer. Overall survival (OS) and disease-free survival (DFS) were assessed after 5 years follow-up. TMA slides were stained with monoclonal antibodies against ROR1, HER3, ER, PR, Ki67, P53, HER2 and CK5/6 using IHC and correlation between the investigated tumor markers and the clinico-pathological parameters of patients were analyzed. RESULTS: Our results showed a significant correlation between ROR1 and ER, PR, HER3, and CK5/6 expression. Additionally, there was a significant correlation between HER3 and ER, PR, HER2, and Ki67 expression. Ki67 was also correlated with HER2 and P53 expression. HER3 expression was significantly correlated with tumor stage, lymph node metastasis, perineural invasion, and multifocal tumors. Furthermore, ROR1 expression was significantly associated with tumor metastasis, lympho-vascular invasion, and perineural invasion. While HER2-HER3 coexpression was significantly associated with poor OS, HER3-ROR1 coexpression was associated with lymph node invasion, lymph node metastasis, and distant metastasis. CONCLUSION: ROR1 and HER3 displayed significant association with different clinic-pathological features and in addition to the other tumor biomarkers could be considered as diagnostic and prognostic biomarkers in breast cancer patients.

Healthy Male Individuals Possess Higher Plasma HER-2 Level than Females.

Considering HER2 as one of the well-known biomarkers in the cancer field, and published articles regarding serum levels of HER2, in this paper we tried to highlight the issue that most studies don't stratify the HER-2 concentration of individuals in terms of gender. In this brief survey, healthy individuals with no prior non-communicable diseases were categorized as males (n=34) and females (n=43), and all samples were evaluated for plasma HER-2 levels at once. Surprisingly, the plasma level of HER-2 of healthy male individuals (mean= 2.28 ± 0.21 ng/mL) was significantly (P<0.0001) higher than the plasma level of HER-2 of healthy females (mean: 0.06 ± 0.09 ng/mL), with no overlap. Therefore, we suggest that more studies are required to re-check the cutoff values for HER-2 plasma levels based on gender since the clinical implications of a unique HER-2 cutoff for both genders may be seriously concerning.

Up-regulation of FOXN3-AS1 in invasive ductal carcinoma of breast cancer patients.

Oncogenic and tumor-suppressive roles of long non-coding RNA make them an appropriate target for expression analysis in cancer studies. In this study, we selected two lncRNAs (EMX2OS and FOXN3-AS1) that are resided near the GWAS-identified SNPs for breast cancer (rs2901157 and rs141061110). These transcripts have been identified in different cancer types as either oncogenes or tumor suppressors. In the present investigation, we aimed to quantify the expression level of EMX2OS and FOXN3-AS1 in 44 breast cancer samples and normal adjacent tissues (ANCTs). The FOXN3-AS1 expression level was significantly increased in breast cancer samples compared with ANCTs (P value = 0.02), Also its amounts could distinguish two sets of samples with an accuracy of 70% (P value = 0.009). We have found an association between FOXN3-AS1 expression and tumor size (P value = 0.02). On the other hand, no significant differences were found in the EMX2OS expression level between two sets of samples (P value = 0.44); however, EMX2OS expression level has a significant association with the age of the patients (P value = 0.03). According to our result, FOXN3-AS1 can be demonstrated as a probable diagnostic marker in breast cancer so we suggest further functional studies to find the precise role of these lncRNAs in breast cancer progression.

Down-Regulation of miR-200c and Up-Regulation of miR-30c Target both Stemness and Metastasis Genes in Breast Cancer.

OBJECTIVE: microRNAs (miRNAs) play important role in progression of tumorigenesis. They can target self-renewal and epithelial-mesenchymal transition (EMT) abilities in tumor cells, especially in cancer stem cells (CSCs). The objective of this study was to implement data mining to identify important miRNAs for targeting both self-renewal and EMT. We also aimed to evaluate these factors in mammospheres as model of breast cancer stem cells (BCSCs) and metastatic tumor tissues. MATERIALS AND METHODS: In this experimental study, mammospheres were derived from MCF-7 cells and characterized for the CSCs properties. Then expression pattern of the selected miRNAs in spheroids were evaluated, using the breast tumor cells obtained from seven patients. Correlation of miRNAs with self-renewal and EMT candidate genes were assessed in mammospheres and metastatic tumors. RESULTS: The results showed that mammospheres represented more colonogenic and spheroid formation potential than MCF-7 cells (P<0.05). Additionally, they had enhanced migration and invasive capabilities. Our computational analyses determined that miR-200c and miR-30c could be candidates for targeting both stemness and EMT pathways. Expression level of miR-200c was reduced, while miR-30c expression level was enhanced in mammospheres, similar to the breast tumor tissues isolated from three patients with grade II/III who received neo-adjuvant treatment. Expression level of putative stem cell markers (OCT4, SOX2, c-MYC) and EMT-related genes (SNAIL1, CDH2, TWIST1/2) were also significantly increased in mammospheres and three indicated patients (P<0.05). CONCLUSION: Simultaneous down-regulation and up-regulation of respectively miR-200c and miR-30c might be signature of BCSC enrichment in patients post neo-adjuvant therapy. Therefore, targeting both miR-200c and miR-30c could be useful for developing new therapeutic approaches, against BCSCs.

Significant random signatures reveals new biomarker for breast cancer.

BACKGROUND: In 2012, Venet et al. proposed that at least in the case of breast cancer, most published signatures are not significantly more associated with outcome than randomly generated signatures. They suggested that nominal p-value is not a good estimator to show the significance of a signature. Therefore, one can reasonably postulate that some information might be present in such significant random signatures. METHODS: In this research, first we show that, using an empirical p-value, these published signatures are more significant than their nominal p-values. In other words, the proposed empirical p-value can be considered as a complimentary criterion for nominal p-value to distinguish random signatures from significant ones. Secondly, we develop a novel computational method to extract information that are embedded within significant random signatures. In our method, a score is assigned to each gene based on the number of times it appears in significant random signatures. Then, these scores are diffused through a protein-protein interaction network and a permutation procedure is used to determine the genes with significant scores. The genes with significant scores are considered as the set of significant genes. RESULTS: First, we applied our method on the breast cancer dataset NKI to achieve a set of significant genes in breast cancer considering significant random signatures. Secondly, prognostic performance of the computed set of significant genes is evaluated using DMFS and RFS datasets. We have observed that the top ranked genes from this set can successfully separate patients with poor prognosis from those with good prognosis. Finally, we investigated the expression pattern of TAT, the first gene reported in our set, in malignant breast cancer vs. adjacent normal tissue and mammospheres. CONCLUSION: Applying the method, we found a set of significant genes in breast cancer, including TAT, a gene that has never been reported as an important gene in breast cancer. Our results show that the expression of TAT is repressed in tumors suggesting that this gene could act as a tumor suppressor in breast cancer and could be used as a new biomarker.

Assessment of the expression pattern of mTOR-associated lncRNAs and their genomic variants in the patients with breast cancer.

The mechanistic target of rapamycin (mTOR) is a fundamental component of a signaling pathway that is involved in the pathogenesis of breast cancer via different mechanisms. This pathway is functionally linked with a number of small nucleolar RNA host genes (SNHGs). In the present project, we have searched for the expression quantitative trait loci (eQTLs) within SNHGs that are possibly involved in the pathogenesis of breast cancer. Following this in silico step, we have assessed expression levels of mTOR and four SNHGs in malignant and nonmalignant samples obtained from 80 patients with breast cancer. We also genotyped rs4615861 of SNHG3 and rs3087978 of SNHG5 in the peripheral blood of patients. SNHG12 expression was not detected in any of the assessed malignant or nonmalignant tissues. So this gene was excluded from further steps. Expression of mTOR and other three long noncoding RNAs (lncRNAs) were significantly increased in the malignant tissues compared with the nonmalignant tissues. When classifying patients into down-/upregulation categorized based on the transcript levels of each gene in malignant tissue versus nonmalignant tissues, we noticed associations between expression of SNHG1 and stage (p = 0.03), expression of SNHG5 and grade (p = 0.05), as well as between expression of SNHG3 and history of oral contraceptive use (p = 0.04). We also detected higher levels of SNHG3 expression in estrogen receptor/progesterone receptor (ER/PR) negative tumors compared with the ER/PR positive tumors (p = 0.003 and p = 0.01, respectively). Moreover, there was a trend toward higher expression of this lncRNA in HER2-positive tumors compared with the HER2-negative ones (p = 0.07). Combination of transcript levels of all genes could differentiate malignant tissues from nonmalignant tissues with the diagnostic power of 69% (p = 0.0001). The rs3087978 was associated with the expression of mTOR in malignant tissues in a way that TT and TG genotypes were associated with the higher and lower levels of expressions, respectively (p = 0.01). The current study underscores the significance of SNHGs in the pathogenesis of breast cancer.

Expression analysis of Inhibitor Of DNA Binding 1 (ID-1) gene in breast cancer.

BACKGROUND: ID-1 gene codes for a helix-loop-helix (HLH) protein that inhibits the DNA binding and transcriptional activation function of these proteins. METHODS: We analyzed ID-1 expression in microarray and RNA Sequencing databases as well as 61 breast cancer tissues compared with adjacent non-cancerous tissues (ANCTs). RESULTS: Expression analysis of ID-1 gene in two microarray datasets and RNA sequencing data showed down-regulation of ID-1 in tumoral tissues compared with normal tissues. However, ID-1 expression analysis in tumoral tissues and ANCTs obtained from 61 patients revealed its over-expression in tumoral tissues. A negative association was detected between ID-1 expression levels and ER status. CONCLUSION: ID-1 expression may be implicated in the pathogenesis of breast cancer especially in patient with ER negative status.

Expression analysis of selected miR-206 targets from the transforming growth factor-ß signaling pathway in breast cancer.

Breast cancer as a molecularly heterogeneous malignancy is associated with dysregulation of several signaling pathways, including transforming growth factor-ß (TGF-ß) signaling. On the other hand, several recent studies have demonstrated the role of microRNAs (miRNAs) in breast cancer pathogenesis. In the current study, we performed a computerized search to find miR-206 target genes that are functionally linked to the TGF-ß signaling pathway. We selected LEF1, Smad2, and Snail2 genes to assess their expression in 65 breast cancer samples and their adjacent noncancerous tissues (ANCTs) in correlation with expression levels of miR-206 as well as clinicopathological characteristics of patients. miR-206 was significantly downregulated in (Estrogen receptor) ER-positive breast cancer samples compared with their corresponding ANCTs. Association analysis between expression levels of genes and demographic features of patients showed significant association between expressions of SMAD2 and LEF1 genes and body mass index ( P values of 0.03 and 0.02, respectively). miR-206 low-expression levels were associated with TNM stage, mitotic rate, and lymph node involvement ( P values of 0.02, 0.01, and 0.01 respectively). In addition, SMAD2 high-expression levels were associated with HER2 status ( P = 0.02). Consequently, our data highlight the role of TGF-ß signaling dysregulation in the pathogenesis of breast cancer and warrant further evaluation of miRNAs and messenger RNA coding genes in this pathway to facilitate detection of cancer biomarkers and therapeutic targets.

Assessment of functional variants and expression of long noncoding RNAs in vitamin D receptor signaling in breast cancer.

PURPOSE: Vitamin D receptor (VDR) signaling pathway is implicated in the pathogenesis of breast cancer. PATIENTS AND METHODS: We selected VDR-associated long noncoding RNAs (lncRNAs) through an in silico analysis of available microarray and RNA-sequencing data and assessed their expression in 75 breast tumor samples and their adjacent noncancerous tissues (ANCTs). We also genotyped two functional polymorphisms within VDR gene in all patients. RESULTS: VDR, MALAT1, and LINC00511 were significantly upregulated in tumoral tissues compared with ANCTs (fold change [FC] =1.85, P=0.03; FC =1.54, P=0.04; and FC =4.75, P=0.000, respectively). In patients younger than 55 years, significant associations were found between expression levels of both SNHG16 and LINC00511 genes and nuclear grade (P=0.03), expression of LINC00346 and tubule formation (P=0.01), expression of both SNHG16 and SNHG6 genes and family history of cancer (P=0.01 and 0.03, respectively), as well as expression of VDR and progesterone receptor status (P=0.03). We detected significant correlations between expression levels of VDR and SNHG16 in both tumoral tissues and ANCTs. The TT genotype of FokI polymorphism was associated with the higher expression levels of VDR. FokI variants were associated with expression levels of both MALAT1 and SNHG16 in ANCTs (P=0.01 and 0.03, respectively). CdxII variants were associated with expression levels of SNHG16 in ANCTs. A significant correlation was found between FC values of SNHG16 expression and vitamin D levels. CONCLUSION: The present study provides further evidence for the contribution of VDR signaling and the related lncRNAs in the pathogenesis of breast cancer and introduces some novel lncRNAs as putative molecules in the interactive functional network of VDR signaling in breast cancer.

Expression analysis of AFAP1-AS1 and AFAP1 in breast cancer.

Long non-coding RNAs (lncRNA) constitute a significant percentage of RNAs with no translation to proteins. Their participation in fundamental aspects of cell physiology as well as their dysregulation in a number of pathologic conditions such as cancer have been documented. Among lncRNAs is actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) whose elevated expression levels have been demonstrated in different cancers. In the in the present study we evaluated expression levels of AFAP1-AS1 and its antisense protein coding gene AFAP1 in breast cancer samples compare with adjacent non-cancerous tissues (ANCTs) as well as breast cancer cell lines with special focus on the assessment of the association between their transcript levels and patients' clinicopathological data. AFAP1-AS1 has shown significant up-regulation in both MDA-MB-231 and MCF-7 compared with control sample. AFAP1-AS1 has been shown to be expressed in all of tumor tissues but 76% (39 out of 51) ANCTs. AFAP1 expression was not significantly different between tumor samples and ANCTs. AFAP1-AS1 has been demonstrated to be significantly up-regulated in tumor tissues compared with ANCTs (fold change = 4.65, P= 0.028). No significant correlation has been detected between the levels of these two transcripts in tumor tissues (R=2 0.081) or ANCTs (R=2 0.115). No significant associations have been found between expression levels of these genes and patients' characteristics. However, both genes were significantly down-regulated in Ki-67 negative tumor samples. The observed up-regulation of AFAP1-AS1 in tumor samples compared with ANCTs implies its involvement in breast cancer pathogenesis and potentiates it as a biomarker or therapeutic target.

Expression Analysis of Long Non-Coding PCAT-1in Breast Cancer.

Background: The prostate cancer-associated non-coding RNA transcript 1 (PCAT-1) is a newly identified long non- coding RNA whose participation in tumorigenesis of a variety of cancers has been observed. In the present study, we aimed at analysis of its expression in breast cancer patients. Materials and Methods: The expression of PCAT-1 was assessed using real-time reverse transcription polymerase chain reaction in tumor samples obtained from 47newly diagnosed breast cancer patients as well as their corresponding adjacent non-cancerous tissues (ANCTs). Results: We detected significant over-expression of PCAT-1 in 12/47 (25.5%) of tumoral tissues compared with their corresponding ANCTs. However, no significant association has been found between the levels of PCAT-1 transcripts and patients' clinical data such as tumor size, stage, grade, estrogen and progesterone receptors or Her2/neu status. Conclusion: PCAT-1 is possibly involved in the pathogenesis of fraction of breast cancers. Future studies are needed to evaluate its precise function in breast cancer.

Expression analysis of long non-coding ATB and its putative target in breast cancer.

BACKGROUND: A long noncoding RNA (lncRNA) activated by transforming growth factor (TGF)-ß (lncRNA-ATB) has been recently shown to promote the invasion-metastasis cascade in various types of cancers via upregulation of some targets including ZEB1. OBJECTIVES: The aim of the present study was to elucidate the expression of lncRNA-ATB and ZEB in breast cancer patients. METHODS: The expression of these genes was evaluated by real-time reverse transcription polymerase chain reaction in tumor samples form 50 newly diagnosed breast cancer patients as well as their corresponding adjacent non-cancerous tissues (ANCTs). Patients were divided into subsequent groups according to the median lncRNA-ATB expression. RESULTS: LncRNA-ATB has been shown to be downregulated in about two third of tumor samples compared with their ANCTs.A significant association has been found between ZEB1 expression and Ki-67 status. In addition, we demonstrated a correlation between expression of lncRNA-ATB and ZEB1 in tumor samples and not in ANCTs. CONCLUSION: Collectively, out data show downregulation of lncRNA-ATB in a significant number of breast tumor tissues compared with ANCTs and imply that lncRNA-ATB might have distinct roles in the pathogenesis of different cancers or even different subtypes of a certain cancer which should be evaluated in future studies.

Expression Study and Clinical Correlations of MYC and CCAT2 in Breast Cancer Patients

Background: Colon cancer-associated transcript 2 (CCAT2) is a newly recognized lncRNA transcribed from the 8q24 genomic region. It functions as an oncogene in various types of cancers including breast cancer, in which it affects Wnt/ß-catenin pathway. Previous studies have shown a putative interaction between this lncRNA and MYC proto-oncogene. Methods: In the current study, we evaluated the expression of CCAT2 in breast cancer tissues with regards to the expression of its target MYC. In addition, we assessed the relationship between CCAT2 and MYC expression levels in tumor tissues and the clinical prognostic characteristics of breast cancer patients. Results: MYC expression levels were significantly up-regulated in tumor tissues compared with adjacent non-cancerous tissues (ANCTs), while such analysis showed no statistically significant difference between these two tissue types in CCAT2 expression. Starkly increased CCAT2 gene expression levels were found in 12/48 (25%) of cancer tissue samples compared with their corresponding ANCTs. Furthermore, significant inverse correlations were found between CCAT2 expression and stage, as well as lymph node involvement. Besides, a significant inverse correlation was found between the relative MYC expression in tumor tissues compared with their corresponding ANCTs and disease stage. Conclusion: These results highlight the significance of MYC and CCAT2 expressions in the early stages of breast cancer development and suggest a potentially significant role for CCAT2 in a subset of breast cancer patients, which could be applied as a potential therapeutic target in these patients.

Comparative expression analysis of hypoxia-inducible factor-alpha and its natural occurring antisense in breast cancer tissues and adjacent noncancerous tissues.

Hypoxia-inducible factors (HIFs) have been shown to be upregulated in tumor tissues and linked with tumor progression and metastasis in breast cancer. Among regulatory mechanisms for HIF expression is a natural occurring antisense named aHIF, which has been shown to be overexpressed in breast cancer and influence the level of the HIF-1α transcript. In the present study, we analyzed the expression of HIF-1α and aHIF in breast cancer tissues versus adjacent noncancer tissues (ANCTs) in relation with the clinical and biological behavior of the tumors. aHIF has been shown to be expressed in 67.4% of invasive ductal carcinoma samples, while none of ANCTs showed its expression. HIF-1α has been expressed in all of tumors and 90% of ANCTs. Comparison of HIF-1α expression level between tumor and ANCT tissues showed a total upregulation in tumor samples. No statistically significant association has been found between the level of HIF-1α expression in tumor samples and clinicopathologic and demographic characteristics such as age, tumor size, estrogen receptor status, progesterone receptor status, HER2/neu expression level, lymph node status, histological grade, and stage except for a weak correlation between HIF-1α expression and Ki-67 status. Besides, we could not detect any significant correlation between relative expression of HIF-1α and aHIF in tumor samples. Collectively, these data suggest that aHIF overexpression can be used as a potential biomarker in breast cancer. However, further studies are needed for the evaluation of its mechanism of action in regulation of HIF-1α expression in different pathological conditions. HIF-1α overexpression results in the upregulation of several genes that participated in cancer-associated pathways such as proliferation, angiogenesis, and glucose metabolism. We showed that HIF-1α is upregulated in breast tumor samples compared with adjacent noncancerous tissues. Its expression has been associated with Ki-67 status. Its natural occurring antisense is only expressed in tumor tissues. Thus, it can be used as a potential biomarker in breast cancer.

Expression analysis of four long noncoding RNAs in breast cancer.

Breast cancer is a molecularly heterogeneous disease which necessitates a search for markers to provide a more specific classification of this disorder. Long noncoding RNAs as the important subset of noncoding transcripts have been shown to be involved in tumorigenic processes. So, they may be used as markers for early detection of cancer and evaluation of cancer prognosis. In addition, they can be applied as therapeutic targets. In this study, we analyzed expression of four long noncoding RNAs (lncRNAs) namely SOX2OT, PTPRG-AS1, ANRASSF1, and ANRIL in 38 breast cancer tissues and their adjacent noncancerous tissues (ANCTs). ANRASSF1 expression was not detected in any noncancerous tissue. All lncRNAs showed significant overexpression in tumor tissues compared with ANCTs. No association was found between gene expressions and individual clinical data such as tumor stage, grade, size and hormone receptor status except for ANRASSF1 expression and Her2/neu status. In addition, ANRASSF1 and ANRIL expressions were significantly higher in triple negative samples. This study suggests a putative role for these lncRNAs in breast cancer and implies that they can be used as potential cancer biomarkers.

Upregulation of RHOXF2 and ODF4 Expression in Breast Cancer Tissues.

OBJECTIVE: During the past decade, the importance of biomarker discovery has been highlighted in many aspects of cancer research. Biomarkers may have a role in early detection of cancer, prognosis and survival evaluation as well as drug response. Cancer-testis antigens (CTAs) have gained attention as cancer biomarkers because of their expression in a wide variety of tumors and restricted expression in testis. The aim of this study was to find putative biomarkers for breast cancer. MATERIALS AND METHODS: In this applied-descriptive study, the expression of 4 CTAs, namely acrosin binding protein (ACRBP), outer dense fiber 4 (ODF4), Rhox homeobox family member 2 (RHOXF2) and spermatogenesis associated 19 (SPATA19) were ana- lyzed at the transcript level in two breast cancer lines (MCF-7 and MDA-MB-231), 40 invasive ductal carcinoma samples and their adjacent normal tissues as well as 10 fibroadenoma samples by means of quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: All four genes were expressed in both cell lines. Expression of ODF4 and RH- OXF2 was detected in 62.5% and 60% of breast cancer tissues but in 22.5 and 17.5% of normal tissues examined respectively. The expression of both RHOXF2 and ODF4 was upregulated in cancerous tissues compared with their normal adjacent tissues by 3.31 and 2.96-fold respectively. The expression of both genes was correlated with HER2/neu overexpression. RHOXF2 expression but not ODF4 was correlated with higher stages of tumors. However, no significant association was seen between expression patterns and estrogen and progesterone receptors status. CONCLUSION: ODF4 and RHOXF2 are proposed as putative breast cancer biomarkers at the transcript level. However, their expression at protein level should be evaluated in future studies.

Expression analysis of two cancer-testis genes, FBXO39 and TDRD4, in breast cancer tissues and cell lines.

Breast cancer accounts for one third of new cancer cases among women. The need for biomarkers for early detection is the stimulus to researchers to evaluate altered expression of genes in tumours. Cancer-testis (CT) genes are a group with limited expression in normal tissues except testis but up-regulation in a wide variety of cancers. We here evaluated expression of two CT genes named FBXO39 and TDRD4 in 32 invasive ductal carcinoma samples, 10 fibroadenomas and 6 normal breast tissue samples, in addition to two breast cancer cell lines, MCF-7 and MDA-MB-231, by the means of quantitative real time RT-PCR. FBXO39 showed significant up-regulation in invasive ductal carcinoma samples in comparison with normal samples. It also was expressed in both cell lines and after RHOXF1 gene knock down it was down-regulated in MCF-7 but up-regulated in the MDA-MB-231 cell line. TDRD4 was not expressed in the MCF-7 cell line and any of the tissue samples except testis. However, it was expressed in MDA-MB-231 and was up-regulated after RHOXF1 gene knock down. Our results show that FBXO39 but not TDRD4 can be used for cancer detection and if proved to be immunogenic, might be a putative candidate for breast cancer immunotherapy.