Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Targeting CD82/KAI1 for Precision Therapeutics in Surmounting Metastatic Potential in Breast Cancer.

Metastasis is the main cause of mortality in breast cancer patients. There is an unmet need to develop therapies that can impede metastatic spread. Precision oncology has shown great promise for the treatment of cancers, as the therapeutic approach is tailored to a specific group of patients who are likely to benefit from the treatment, rather than the traditional approach of "one size fits all". CD82, also known as KAI1, a glycoprotein belonging to the tetraspanin family and an established metastasis suppressor, could potentially be exploited to hinder metastases in breast cancer. This review explores the prospect of targeting CD82 as an innovative therapeutic approach in precision medicine for breast cancer patients, with the goal of preventing cancer progression and metastasis. Such an approach would entail the selection of a subset of breast cancer patients with low levels of CD82, and instituting an appropriate treatment scheme tailored towards restoring the levels of CD82 in this group of patients. Proposed precision treatment regimens include current modalities of treating breast cancer, in combination with either clinically approved drugs that could restore the levels of CD82, CD82 peptide mimics or non-coding RNA-based therapeutics.

Genetic differences between benign phyllodes tumors and fibroadenomas revealed through targeted next generation sequencing.

Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.

Gold nanoparticles induce serum amyloid A 1-Toll-like receptor 2 mediated NF-kB signaling in lung cells in vitro.

Gold nanoparticles (AuNPs) have emerging applications in biomedicine and the industry. Exposure to AuNPs has previously been shown to alter the transcriptional activity of nuclear factor kappa B (NF-kB), which is known to mediate physiological and pathological processes. This study seeks to provide mechanistic insights into AuNP-induced NF-kB activation in Small Airway Epithelial Cells (SAECs) in vitro. Increased NF-kB transcriptional activity (quantified by the luciferase reporter assay) was observed in AuNP-treated SAECs. Transcriptomic analysis revealed differential expression of 42 genes, which regulate functional processes that include cellular response to stimulus, chemicals and stress as well as immune response. Notably, the gene expression of serum amyloid A1 (SAA1), an acute phase protein and Toll-like receptor 2 (TLR2) were found to be up-regulated. As TLR2 is known to be a functional receptor of SAA1, a co-immunoprecipitation assay was performed. SAA1 was observed to be co-immunoprecipitated with the TLR2 protein and this protein-protein interaction was further supported by in silico computer based protein modeling. The present study suggests that AuNPs may potentially induce SAA1-TLR2-mediated NF-kB transcription factor activation in lung epithelial cells, highlighting that nano-bio interactions could result in biological effects that may affect cells.

Size and heterologous elements predict metastases in malignant phyllodes tumours of the breast.

Phyllodes tumours (PTs) of the breast are uncommon fibroepithelial neoplasms comprising 0.3-1.0% of all primary breast tumours. The majority of PTs are benign and generally well managed with surgery. However, malignant PTs, and occasionally borderline PTs, can behave in a clinically aggressive manner by metastasizing to distant organs. Although distant metastasis is rare, the prognosis of patients with metastasis is dismal as many are unresponsive to standard chemotherapy and the risk of death is high. In this study, we correlated clinicopathological parameters to survival outcomes in a cohort of patients diagnosed with malignant PTs in our institution. The study cohort comprised 83 cases of malignant PTs diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 1994 to 2015. Clinicopathological features and follow-up were obtained from hospital records. Metastasis-free survival (MFS) and overall survival (OS) were estimated with the Kaplan-Meier method and compared between groups using the log-rank test. Cox regression was carried out to identify factors predictive for metastasis. Mean and median age of patients was 48 years (range 21-71 years). Tumour size measured from 30 to 220 mm (mean 90 mm, median 77 mm). Follow-up data was available for 68 patients. Mean and median follow-up was 90 and 57 months, respectively, with a maximum of 291 months. Distant metastasis occurred in 16 out of 68 patients (23.5%). The most common site of metastasis was the lung. Malignant heterologous elements were observed in 16 (19.3%) cases. Individual clinicopathological parameters had no impact on outcome. On Kaplan-Meier analysis, women with large tumours and presence of malignant heterologous elements showed trends for poorer MFS (p = 0.217 and p = 0.566, respectively). However, the combination of large tumours (≥ 90 mm) containing malignant heterologous elements disclosed significantly worse MFS (p = 0.043) and a trend for poorer OS (p = 0.238). On multivariate analysis, large tumours harbouring malignant heterologous elements independently predicted metastasis (95% CI 1.041-12.517, HR 2.434, p = 0.049). Our study demonstrates that tumour size and presence of malignant heterologous elements predicted metastasis in malignant PTs. Further work needs to be done in determining if protein biomarkers and genomic aberrations are able to additionally refine metastatic risk and define therapeutic targets.

Silencing HS6ST3 inhibits growth and progression of breast cancer cells through suppressing IGF1R and inducing XAF1.

Heparan sulfate 6-O-sulfation is biologically edited by 6-O-sulfotransferases (HS6STs) within heparan sulfate chains. Three isoforms of HS6ST have been identified. These enzymes are found to be differentially expressed in a variety of tissues. Recently, several studies have shown that dysregulation of 6-O-sulfotransferases could be involved in tumorigenesis of several cancers. This study aimed to analyze the expression and function of HS6ST3 in breast cancer. HS6ST3 was found up-regulated in T47D, MCF7 and MDA-MB231 breast cancer cell lines. HS6ST3 was then silenced in T47D and MCF7 using siRNA. Silencing HS6ST3 diminished tumor cell growth, migration and invasion, but enhanced cell adhesion and apoptosis in breast cancer. Gene microarray analysis revealed that silencing HS6ST3 significantly changed the expression of IGF1R and XAF1 in breast cancer cells. Further functional studies showed that the cellular processes were mediated by IGF1R and XAF1 after silencing HS6ST3 in breast cancer cells. Together these results indicate that HS6ST3 might be involved in the tumorigenesis of breast cancer and it could be a promising target in breast cancer therapy.

Prognostic significance of Claudin 12 in estrogen receptor-negative breast cancer.

AIMS: Altered expression of the Claudin (CLDN) superfamily of tight junction proteins has been reported in breast cancer. The aim of this study was to examine the immunohistochemical expression of CLDN 12 and its prognostic significance in breast cancer tissues. METHODS: Immunohistochemical expression of CLDN 12 was performed on tissue microarrays consisting of 232 cases of breast carcinoma and correlated with clinicopathological features as well as survival of the patients with breast cancer. RESULTS: For the estrogen receptor (ER)-negative subgroup of patients with breast cancer, CLDN 12 expression was shown to be an independent predictor of poor overall survival (HR=2.345; p=0.020) and disease-free survival (HR=2.177; p=0.026) but not for the ER-positive tumours. CONCLUSIONS: The findings suggest that CLDN 12 expression could be clinically useful for predicting the survival of the ER-negative subgroup of patients with breast cancer.

Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer.

Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.

Y-box binding protein 1 is correlated with lymph node metastasis in intestinal-type gastric cancer.

AIMS: Y-box binding protein-1 (YB-1) is known to modulate gene transcription and protein translation, as well as cellular response to drug treatment. The aim of this study is to correlate YB-1 protein expression levels with clinicopathological parameters in intestinal-type gastric cancer tissue samples (as categorized by the Lauren classification) and substantiate the findings with in vitro experimentation. METHODS AND RESULTS: Paraffin-embedded samples from 167 patients with intestinal-type gastric cancer were used for the construction of tissue microarrays (TMAs). TMA slides were immunostained and YB-1 immunoreactivity score was based on the weighted average intensity score. Univariate analysis revealed that YB-1 immunohistochemical expression was correlated significantly with lymph node status (P = 0.054, borderline significance) and perforation (P = 0.043). YB-1 expression was also found to be an independent predictor of lymph node spread by multivariate analysis. Furthermore, siRNA-mediated YB-1 gene knockdown in MKN7 gastric cancer cells (which is known to originate from an intestinal-type gastric cancer tissue) inhibited cell migration (P = 0.0002) and invasion in vitro (P = 0.0129) significantly. CONCLUSION: YB-1 expression is associated with lymph node spread in intestinal-type gastric cancer and is a potential prognostic biomarker in this subtype of gastric cancer.

Syndecan-1 is a potential biomarker for triple-positive breast carcinomas in Asian women with correlation to survival.

INTRODUCTION: While overexpression of syndecan-1 has been associated with aggressive breast cancer in the Caucasian population, the expression pattern of syndecan-1 in Asian women remains unclear. Triple-positive breast carcinoma, in particular, is a unique subtype that has not been extensively studied. We aimed to evaluate the role of syndecan-1 as a potential biomarker and prognostic factor for triple-positive breast carcinoma in Asian women. METHODS: Using immunohistochemistry, staining scores of 61 triple­positive breast carcinoma specimens were correlated with patients' clinicopathological variables such as age, ethnicity, tumour size, histological grade, lymph node status, lymphovascular invasion, associated ductal carcinoma in situ grade, recurrence and overall survival. RESULTS: Syndecan-1 had intense staining scores in triple­positive invasive ductal breast carcinomas when compared to normal breast tissue. On multivariate analysis, syndecan-1 epithelial total percentage and immunoreactivity score showed statistical correlation with survival (p = 0.02). CONCLUSION: The intense staining scores of syndecan-1 and their correlation with overall survival in patients with triple-positive breast carcinoma suggest that syndecan-1 may have a role as a biological and prognostic marker in patients with this specific subtype of breast cancer.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers.

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-ß-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Claudins and gastric carcinogenesis.

Gastric carcinoma arises from aberrant growth of normal gastric mucosa. There is increasing evidence that claudins (CLDNs) may play a critical role in the significant steps of gastric tumorigenesis, from metaplasia to metastasis. The CLDN family which consists of at least 27 member proteins is known to mediate selective permeability in cellular tight junctions. It is now established that CLDNs are differentially altered in gastric cancer and CLDN proteins are believed to play different roles in the growth and progression of gastric cancer.

Silencing the YB-1 gene inhibits cell migration in gastric cancer in vitro.

The Y-Box-Binding Protein-1 (YB-1) is known to regulate the processes of transcription, translation, cellular response to drug treatment and viral infection as well as DNA repair among others. As gastric cancer is a common cancer with a high incidence in countries in Asia, we evaluated the association of YB-1 with the malignant potential of gastric cancer cells in vitro. YB-1 mRNA expression levels were first determined by real-time RT-PCR in two adherent gastric cancer cell lines (viz., MKN7 and NUGC3 gastric cancer cells) and a normal GES-1 gastric epithelial cell line. Poorly differentiated NUGC3 gastric cancer cells were found to have the highest YB-1 gene expression among the adherent cells. YB-1 gene expression was also observed to be higher in non-adherent SNU5 gastric cancer cells compared to more aggressive SNU16 cells. Silencing of the YB-1 gene by siRNA in NUGC3 cells was associated with a significant reduction of the YB-1 protein by more than 55% as verified by Western blot analysis. Down-regulation of YB-1 protein expression was further demonstrated qualitatively by immunocytochemistry and immunofluorescence staining. Silencing of the YB-1 gene induced significant inhibition of cell migration in NUGC3 cells by 60% but did not influence cell invasion. Although epithelial-mesenchymal-transition (EMT) is known to be associated with the migratory phenotype in cancer cells, there was no change in the expression of EMT genes when YB-1 expression was modulated. YB-1 appears to have an integral role in cancer cell migration, a process which is important for gastric cancer metastasis.

Downregulation of ER60 protease inhibits cellular proliferation by inducing G1/S arrest in breast cancer cells in vitro.

ER60 protease, a 58-kDa molecular chaperone in the endoplasmic reticulum, is involved in glycoprotein synthesis. ER60 protease has been reported to be differentially expressed in various cancers including breast carcinoma. This study explored the relationship of ER60 protease with cell proliferation in breast cancer in vitro. ER60 protease expression was first determined in a panel of breast cell lines by real-time RT-PCR and Western blot analysis and found to be most abundantly expressed in T47D breast cancer cells. The ER60 protease gene was then successfully knocked down in T47D breast cancer cells using two different sequences of small-interfering RNA. The silencing efficiencies of siER-1 and siER-2 at 48-hr post-transfection were found to be >80% at the mRNA level with concomitant downregulation of the ER60 protease protein by >60% when compared with control T47D breast cancer cells. Downregulation of ER60 protease was also associated with inhibition of cell proliferation when assessed by the AlamarBlue assay. Cell cycle analysis performed on the siER-1- and siER-2-transfected cells, revealed an increase in G1 phase population and a decrease in the S and G2/M phase populations compared with control cells, implicating G1/S cell cycle arrest. It would appear that ER60 protease is involved in breast tumorigenesis and could therefore be a prospective target for cancer therapeutics.

Mutations in the epidermal growth factor receptor (EGFR) gene in triple negative breast cancer: possible implications for targeted therapy.

INTRODUCTION: Triple negative breast cancer is associated with poorer prognosis and unresponsiveness to endocrine and anti-HER2 directed agents. Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare and exploration of additional targeted therapies is needed. Epidermal growth factor receptor (EGFR) is expressed in triple negative breast cancer and several clinical trials are testing the role of anti-EGFR directed therapy. However, the rate of EGFR mutations is poorly defined. We, therefore, sought to characterize EGFR mutations in triple negative breast cancers. METHODS: Seventy samples were randomly chosen from a cohort of 653 triple negative breast tumours for EGFR mutation analysis. These samples were immunostained for EGFR protein expression and consisted of negatively stained and positively stained cases. DNA was extracted from paraffin blocks and polymerase chain reaction was performed to amplify exon regions 18 to 21 of the EGFR gene. Direct sequencing of the purified PCR products was performed. RESULTS: EGFR mutations were found in 8 of 70 samples (11.4%). Mutations were predominantly exon 19 deletions (4 of 70 samples, 5.7%), which clustered in the region spanning codons 746 to 759 within the kinase domain of EGFR. Two types of exon 19 deletions were seen: a 15 nucleotide deletion (del E746-A750) (2 of 70 samples) and a 24 nucleotide deletion (del S752 - I759) (2 of 70 samples). Other exon 19 mutations observed were the inversion of the complementary strand (1 of 70 samples). Exon 21 mutations included missense substitution, L858R (1 of 70 samples) and T847I (2 of 70 samples). Mutations observed were independent of EGFR protein expression determined by immunohistochemical staining. CONCLUSIONS: This study is among the first to document the presence and estimate the prevalence of EGFR mutations in triple negative breast cancer. These findings have potential implications for the design of clinical trials involving anti-EGFR directed therapy which currently do not select for patients based on presence of activating EGFR mutations, which may hence be underpowered to detect significant benefit in unselected populations. More complete sampling of EGFR mutation status in triple negative breast cancer is needed to determine the true mutation rate.

Differential expression of metallothionein in gastrointestinal stromal tumors and gastric carcinomas.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that account for about 2% of gastric tumors. Metallothioneins (MTs) are multifunctional proteins associated with carcinogenesis and known to be coded by 10 functional MT genes. This study evaluated MT mRNA and protein expression in GISTs and compared the expression levels with gastric carcinomas. An immunohistochemical study of MT protein expression was performed in 15 GISTs (specifically located in the stomach) and 38 early stage gastric carcinomas. The percentage of cells stained and intensity of staining were determined. MT-2A mRNA expression was investigated in 6 GISTs and 6 early stage gastric carcinoma patients. All GISTs displayed positive nuclear immunostaining, with most GISTs having predominantly mildly stained nuclei (93.3%). On the other hand, 37 out of 38 gastric carcinoma cases were positively stained for nuclear MT with 24 cases (63.2%) exhibiting predominantly mild nuclear staining, 7 cases (18.4%) moderate nuclear staining, and 6 cases (15.8%) strong nuclear staining. Nuclear MT expression was found to be significantly lower in GIST samples when compared with gastric carcinoma tissues based on the percentage stained and immunoreactive score. We then established that the MT-2A gene transcript was the most abundant MT isoform in MKN28 gastric cancer cells and analyzed its expression in GIST and gastric carcinoma tissues. We found that GISTs had significantly lower MT-2A mRNA levels than gastric carcinoma tissues. Lower MT-2A gene expression and nuclear MT protein expression in GISTs when compared with gastric carcinomas may reflect their different underlying biology and divergent histogenesis.

Expression of heparan sulfate in gastric carcinoma and its correlation with clinicopathological features and patient survival.

AIM: To determine the expression pattern and prognostic value of heparan sulfate in gastric cancer. METHOD: The 10E4 antiheparan sulfate monoclonal antibody was used to examine the expression pattern of heparan sulfate in tissue microarrays consisting of 162 cases of gastric carcinoma by immunohistochemistry. The immunoreactivities of both epithelial and stromal components of the specimens were examined and analysed statistically for significant associations with clinicopathological parameters, including histological grade of the tumour, extent of cancer infiltration and presence of lymph-node metastases, lymphovascular invasion, perineural invasion, perforation of gastric wall and stromal reaction. The potential use of heparan sulfate as a predictive factor for patient survival was also evaluated. RESULTS: Reduced expression of heparan sulfate in the epithelial component was associated with higher histological grades of gastric cancer as well as the presence of more extensive tumour infiltration. Furthermore, this decrease in heparan sulfate expression was found to be predictive of reduced patient survival after tumour recurrence. CONCLUSION: The data suggest that heparan sulfate may play an important role in regulating the biology of gastric cancer, and that it may be a useful prognostic marker of this tumour.

Clinicopathological significance of calreticulin in breast invasive ductal carcinoma.

Calreticulin is a chaperone protein located in the lumen of the endoplasmic reticulum. The association of calreticulin with pathological conditions such as autoimmune disorders and certain types of cancer have been reported. However, little is known about its role in the pathogenesis of breast cancer. The aim of this study was to determine the expression of calreticulin in vitro and correlate its expression levels in breast cancer tissue samples with clinicopathological parameters. Calreticulin expression was evaluated in MCF-7 and MDA-MB-231 breast cancer cells by real-time RT-PCR, Western blot, immunohistochemistry, and immunofluorescence staining. Patient tissue microarrays were constructed from 228 breast cancer specimens for immunohistochemical analysis. The in vitro study showed a higher calreticulin expression in more aggressive MDA-MB-231 cells as compared with MCF-7 cells at both mRNA and protein levels. In all, 227 out of 228 breast cancer samples exhibited calreticulin staining in at least 5% of the cancer cells. Calreticulin immunostaining was observed to be localized to the cytoplasm of the cancer cells. Regression analysis of calreticulin immunostaining in the tissue microarrays revealed that its expression was positively correlated to logarithm of (log) tumor size (P=0.046) and development of distant metastasis (P=0.017). Multivariate analysis confirmed calreticulin expression as an independent predictor of log tumor size and occurrence of distant metastasis. The data suggest that calreticulin expression is associated with more advanced tumors and is a potential prognostic biomarker.

Y-box binding protein 1 is up-regulated in proliferative breast cancer and its inhibition deregulates the cell cycle.

The Y-box-binding protein 1 (YB-1), a member of the cold-shock domain RNA-and DNA-binding protein family, has pleiotropic functions such as regulation of the cell cycle. The aim of this study was to evaluate if YB-1 is a proliferative marker in breast cancer and elucidate potential downstream targets involved in YB-1-mediated cell cycle regulation using RNA interference technology. YB-1 protein expression was evaluated in tissue microarrays of 131 breast invasive ductal carcinomas by immunohistochemistry, while the YB-1 gene expression profile was evaluated in the T-47D, MDA-MB-231, ZR-75-1 and MCF7 breast cancer cell lines. Silencing of the YB-1 gene in T-47D breast cancer cells was performed using siRNA and the effects of down-regulation of YB-1 on cell growth and regulation of the cell cycle were ascertained. A focused panel of 84 genes involved in cell cycle progression was also examined. In tissue microarrays, YB-1 expression was shown to be associated with proliferating cell nuclear antigen (PCNA) immunostaining. siRNA-mediated silencing of the YB-1 gene inhibited cell proliferation and induced G1 phase cell cycle arrest in T-47D breast cancer cells. Knockdown of the YB-1 gene induced up-regulation of two genes which contribute to G1-arrest (RAD9A and CDKN3 genes) and down-regulation of ten genes associated with positive regulation of the cell cycle (SKP2, SUMO1, ANAPC4, CCNB1, CKS2, MNAT1, CDC20, RBBP8, KPNA2 and CCNC genes). The data obtained from the tissue microarrays and cell lines provide evidence that YB-1 is a reliable marker of cell proliferation and possibly a potential molecular target in breast cancer therapy.

Silencing the Metallothionein-2A gene induces entosis in adherent MCF-7 breast cancer cells.

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage-dependent MCF-7 cells internalizing neighboring epithelial cells (entosis) after siRNA-mediated silencing of the Metallothionein-2A (MT-2A) gene. MTs belong to a family of low-molecular weight proteins, which bind metal ions endogenously and its over-expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF-7 breast cancer cells by silencing the MT-2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell-in-cell cytostructure after MT-2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT-2A gene silencing.