Association of Notch pathway down-regulation with Triple Negative/Basal-like breast carcinomas and high tumor-infiltrating FOXP3+ Tregs.

T regulatory cells (Tregs) are a lineage of lymphocytes involved in immune response suppression that are characterized by the expression of the forkhead box P3 (FOXP3) transcription factor. Notch pathway regulates FOXP3 transcription in Tregs, but its role in breast cancer is unknown. We aimed at studying whether Notch pathway regulates FOXP3 expression and Tregs content in breast cancer, and its association with luminal breast carcinomas. We analyzed by quantitative Real-Time PCR the mRNA levels of FOXP3, Notch pathway genes (Notch1, Notch2, Notch4 and Jagged1) and STAT3 in a series of 152 breast carcinomas including hormone receptor-positive and -negative phenotypes (luminal and Triple Negative/Basal-like). We also studied the protein expression of Notch1, STAT3 and FOXP3 by immunohistochemistry. High FOXP3 mRNA levels correlated with larger tumor size (p=0.010), histological grade 3 (p=0.008) and positive lymph-node status (p=0.031). Also, low levels of Notch pathway genes mRNA correlated with poor prognostic factors such as larger tumor size, positive lymph-node status, tumor phenotype and infiltrating tumor Tregs. A survival analysis for the patients showed that large tumor size, histological grade 3, vascular invasion, infiltrating Tregs and low Notch1 mRNA expression were significantly associated with a decreased patients' overall survival (p≤0.05). On a multivariate analysis, high Tregs content (HR=3.00, 95% CI 1.04-8.90, p=0.042) and low Notch1 mRNA levels (HR=3.33, 95% CI 1.02-10.86, p=0.046) were independent markers for overall survival. Our results support that the Notch pathway up-regulation promotes luminal breast carcinomas, whereas down-regulation correlates with the expression of FOXP3, favors tumor Tregs infiltration and associates with Triple Negative/Basal-like tumors.

FOXA2 mRNA expression is associated with relapse in patients with Triple-Negative/Basal-like breast carcinoma.

The FOXA family of transcription factors regulates chromatin structure and gene expression especially during embryonic development. In normal breast tissue FOXA1 acts throughout mammary development; whereas in breast carcinoma its expression promotes luminal phenotype and correlates with good prognosis. However, the role of FOXA2 has not been previously studied in breast cancer. Our purpose was to analyze the expression of FOXA2 in breast cancer cells, to explore its role in breast cancer stem cells, and to correlate its mRNA expression with clinicopathological features and outcome in a series of patients diagnosed with breast carcinoma. We analyzed FOXA2 mRNA expression in a retrospective cohort of 230 breast cancer patients and in cell lines. We also knocked down FOXA2 mRNA expression by siRNA to determine the impact on cell proliferation and mammospheres formation using a cancer stem cells culture assay. In vitro studies demonstrated higher FOXA2 mRNA expression in Triple-Negative/Basal-like cells. Further, when it was knocked down, cells decreased proliferation and its capability of forming mammospheres. Similarly, FOXA2 mRNA expression was detected in 10% (23/230) of the tumors, especially in Triple-Negative/Basal-like phenotype (p < 0.001, Fisher's test). Patients whose tumors expressed FOXA2 had increased relapses (59 vs. 79%, p = 0.024, log-rank test) that revealed an independent prognostic value (HR = 3.29, C.I.95% = 1.45-7.45, p = 0.004, Cox regression). Our results suggest that FOXA2 promotes cell proliferation, maintains cancer stem cells, favors the development of Triple-Negative/Basal-like tumors, and is associated with increase relapses.

Detecting zoonotic Influenza A using QIAstat-Dx Respiratory SARS-CoV-2 panel for pandemic preparedness.

Recent reports from the World Health Organization regarding Influenza A cases of zoonotic origin in humans (H1v and H9N2) and publications describing emergence swine Influenza A cases in humans together with "G4" Eurasian avian-like H1N1 Influenza A virus have drawn global attention to Influenza A pandemic threat. Additionally, the current COVID-19 epidemic has stressed the importance of surveillance and preparedness to prevent potential outbreaks. One feature of the QIAstat-Dx Respiratory SARS-CoV-2 panel is the double target approach for Influenza A detection of seasonal strains affecting humans using a generic Influenza A assay plus the three specific human subtype assays. This work explores the potential use of this double target approach in the QIAstat-Dx Respiratory SARS-Co-V-2 Panel as a tool to detect zoonotic Influenza A strains. A set of recently recorded H9 and H1 spillover strains and the G4 EA Influenza A strains as example of recent zoonotic Flu A strains were subjected to detection prediction with QIAstat-Dx Respiratory SARS-CoV-2 Panel using commercial synthetic dsDNA sequences. In addition, a large set of available commercial human and non-human influenza A strains were also tested using QIAstat-Dx Respiratory SARS-CoV-2 Panel for a better understanding of detection and discrimination of Influenza A strains. Results show that QIAstat-Dx Respiratory SARS-CoV-2 Panel generic Influenza A assay detects all the recently recorded H9, H5 and H1 zoonotic spillover strains and all the G4 EA Influenza A strains. Additionally, these strains yielded negative results for the three-human seasonal IAV (H1, H3 and H1N1 pandemic) assays. Additional non-human strains corroborated those results of Flu A detection with no subtype discrimination, whereas human Influenza strains were positively discriminated. These results indicate that QIAstat-Dx Respiratory SARS-CoV-2 Panel could be a useful tool to diagnose zoonotic Influenza A strains and differentiate them from the seasonal strains commonly affecting humans.

ID1 and ID4 Are Biomarkers of Tumor Aggressiveness and Poor Outcome in Immunophenotypes of Breast Cancer.

Inhibitor of differentiation (ID) proteins are a family of transcription factors that contribute to maintaining proliferation during embryogenesis as they avoid cell differentiation. Afterward, their expression is mainly silenced, but their reactivation and contribution to tumor development have been suggested. In breast cancer (BC), the overexpression of ID1 has been previously described. However, whether the remaining ID genes have a specific role in this neoplasia is still unclear. We studied the mRNA expression of all ID genes by q RT-PCR in BC cell lines and 307 breast carcinomas, including all BC subtypes. Our results showed that ID genes are highly expressed in all cell lines tested. However, ID4 presented higher expression in BC cell lines compared to a healthy breast epithelium cell line. In accordance, ID1 and ID4 were predominantly overexpressed in Triple-Negative and HER2-enriched samples. Moreover, high levels of both genes were associated with larger tumor size, histological grade 3, necrosis and vascular invasion, and poorer patients' outcomes. In conclusion, ID1 and ID4 may act as biomarkers of tumor aggressiveness and worse prognosis in breast cancer, and they could be used as potential targets for new treatments discover.

Hedgehog gene expression patterns among intrinsic subtypes of breast cancer: Prognostic relevance.

PURPOSE: Hedgehog (Hh) signaling is a crucial developmental regulatory pathway recognized as a primary oncogenesis driver in various human cancers. However, its role in breast carcinoma (BC) has been underexplored. METHODS: We analyzed the expression of several Hh associated genes in a clinical series and breast cancer cell lines. We included 193 BC stratified according to intrinsic immunophenotypes. Gene expression profiling ofBOC, PTCH, SMO, GLI1, GLI2, and GLI3 was performed by qRT-PCR. Results were correlated with clinical-pathological variables and outcome. RESULTS: We observed expression ofGLI2 in triple-negative/basal-like (TN/BL) and GLI3 in luminal cells. In samples, BOC, GLI1, GLI2, and GLI3 expression correlated significantly with luminal tumors and good prognostic factors. In contrast, PTCH and SMO correlated with TN/BL phenotype and nodal involvement. Patients whose tumors expressed SMO had a poorer outcome, especially those with HER2 phenotype. Positive lymph-node status and high SMO remained independent poor prognostic factors. CONCLUSION: Our results support a differential Hh pathway activation in BC phenotypes.SMO levels stratified patients at risk of recurrence and death in HER2 phenotype, and it showed an independent prognostic value. Therefore, SMO could be a potential therapeutic target for a subset of BC patients.

Statin-induced metabolic reprogramming in head and neck cancer: a biomarker for targeting monocarboxylate transporters.

Prognosis of HPV negative head and neck squamous cell carcinoma (HNSCC) patients remains poor despite surgical and medical advances and inadequacy of predictive and prognostic biomarkers in this type of cancer highlights one of the challenges to successful therapy. Statins, widely used for the treatment of hyperlipidaemia, have been shown to possess anti-tumour effects which were partly attributed to their ability to interfere with metabolic pathways essential in the survival of cancer cells. Here, we have investigated the effect of statins on the metabolic modulation of HNSCC cancers with a vision to predict a personalised anticancer therapy. Although, treatment of tumour-bearing mice with simvastatin did not affect tumour growth, pre-treatment for 2 weeks prior to tumour injection, inhibited tumour growth resulting in strongly increased survival. This was associated with increased expression of the monocarboxylate transporter 1 (MCT1) and a significant reduction in tumour lactate content, suggesting a possible reliance of these tumours on oxidative phosphorylation for survival. Since MCT1 is responsible for the uptake of mitochondrial fuels into the cells, we reasoned that inhibiting it would be beneficial. Interestingly, combination of simvastatin with AZD3965 (MCT1 inhibitor) led to further tumour growth delay as compared to monotherapies, without signs of toxicity. In clinical biopsies, prediagnostic statin therapy was associated with a significantly higher MCT1 expression and was not of prognostic value following conventional chemo-radiotherapy. These findings provide a rationale to investigate the clinical effectiveness of MCT1 inhibition in patients with HNSCC who have been taking lipophilic statins prior to diagnosis.

CD44 induces FOXP3 expression and is related with favorable outcome in breast carcinoma.

We studied the relationship between CD44 and Forkhead box P3 (FOXP3) gene expression in cell lines and breast carcinomas and their association with clinicopathological variables and patient outcome. We assessed messenger RNA (mRNA) expression of CD44 and FOXP3 by quantitative real-time PCR and determined the number of FOXP3+ Tregs by immunohistochemistry in 264 breast cancer specimens. CD44 was stimulated with hyaluronan treatment, and the accompanying changes in FOXP3 mRNA expression in breast cancer cell lines representing breast cancer subtype were assessed. We found that lower CD44 expression correlated with the presence of necrosis, lymph-vascular invasion, grade 3 tumors, and aggressive phenotype (HER2 and basal-like). FOXP3 mRNA correlated positively with CD44 mRNA expression and Treg content. Moreover, stimulation of CD44 expression by hyaluronan in cell lines increased FOXP3 expression, which supports that their regulation is associated. Survival analysis revealed that low CD44 expression is associated with higher frequency of recurrence. Our findings indicate that CD44 has a regulatory role in FOXP3 expression and is associated with good prognostic factors in breast cancer.

Osteopontin Regulates VEGFA and ICAM-1 mRNA Expression in Breast Carcinoma.

OBJECTIVES: To analyze the regulatory role of osteopontin on biomarkers associated with cell survival, invasiveness, and angiogenesis mechanisms in a clinical series and breast cancer cell lines. METHODS: We analyzed by quantitative real-time polymerase chain reaction the messenger RNA (mRNA) expression of osteopontin, Bcl2, intercellular adhesion molecule 1 (ICAM-1), and vascular endothelial growth factor A (VEGFA) in several breast cancer cell lines and in 148 breast carcinomas classified into intrinsic subtypes. RESULTS: We found coexpression of osteopontin, Bcl2, ICAM-1, and VEGFA in triple-negative MDA-MB-468 and MDA-MB-231 cell lines. Furthermore, osteopontin silencing by small interfering RNA inhibited ICAM-1 and VEGFA expression and cell proliferation in MDA-MB-468 cells. In breast cancer specimens, we found a positive correlation between osteopontin, ICAM-1, and VEGFA mRNA expression, especially in triple-negative/basal-like tumors. Among patients with osteopontin-overexpressing tumors, VEGFA remained an independent prognostic indicator for recurrence (hazard ratio, 2.95; 95% confidence interval [CI], 1.48-5.87; P = .002) and death (hazard ratio, 3.25; 95% CI, 1.48-7.11; P = .003) (multivariate analysis, Cox regression). CONCLUSIONS: Our results support that osteopontin regulates ICAM-1 and VEGFA expression mainly in triple-negative/basal-like breast carcinomas, suggesting a relevant role in the pathogenesis and tumor progression of this molecular subtype. Moreover, VEGFA mRNA levels showed an independent prognostic value in patients with breast cancer.

Association of increased osteopontin and splice variant-c mRNA expression with HER2 and triple-negative/basal-like breast carcinomas subtypes and recurrence.

Osteopontin, a secreted phosphoglycoprotein, promotes tumor progression through binding to integrins and CD44 cell receptors. Its overexpression has been correlated with metastasis and adverse outcome in several neoplasms. In breast carcinoma, osteopontin mRNA and its splicing variant-c, a suggested marker for transformed cells, have not been extensively analyzed. Immunohistochemistry was performed in 415 breast carcinomas to examine total osteopontin and osteopontin-c protein distribution. RNA was extracted and retrotranscribed to cDNA from 309 tumors classified into immunophenotypes and in six cell lines representing the breast cancer subtypes. Total osteopontin and osteopontin-c mRNA levels were measured by quantitative RT-polymerase chain reaction. The median fold change of total osteopontin mRNA was higher in HER2-positive (fold-change = 14.7) and triple-negative/basal-like (fold-change = 14.7) tumors, whereas osteopontin-c mRNA was elevated in triple-negative/basal-like subtype (fold-change = 2.8). Total osteopontin levels were increased in SK-BR-3 (HER2-positive) and MDA-MB-468 (triple-negative/basal-like) cell lines. Higher total and osteopontin-c mRNA levels were seen in tumors of high grade, with necrosis, positive nodal status and high Nothingam Prognostic Index score. Disease-free survival was significantly shorter for patients whose tumors overexpressed total osteopontin (67% vs 73%). Moreover, increased osteopontin-c stratified subgroups of patients at higher risk of recurrence among immunophenotypes, especially in triple-negative/basal-like subtype (70% vs 83%). By multivariate analyses for disease-free survival, osteopontin-c emerged as a significant predictor of relapse. In summary, our data showed an association of osteopontin with poor prognostic factors, aggressive subtypes HER2 and triple-negative/basal-like, and higher risk of recurrence.

Association of mammalian target of rapamycin with aggressive type II endometrial carcinomas and poor outcome: a potential target treatment.

The classification of endometrial carcinoma divided into types I and II has shown clinical usefulness. Molecular alterations of PTEN and Wnt/ß-catenin have been identified in this neoplasia. However, the role of mammalian target of rapamycin according to subcellular localization in the pathogenesis of this neoplasia and its prognostic significance are not well defined. We studied the expression of phosphorylated mammalian target of rapamycin, PTEN, and ß-catenin and their relationship with clinicopathologic features, molecular factors (microsatellite instability, mismatch repair, and BRAF genes) and patients' survival in a series of 260 nonconsecutive endometrial carcinomas. Tissue microarrays were manually constructed, and genomic DNA was extracted from paraffin-embedded cylinders (1 mm thick) from preselected tumor areas. The mammalian target of rapamycin in the nuclei (mTORC2; 47%) or cytoplasm (mTORC1; 48%) were seen in type II endometrial carcinoma, the latter also in advanced stages (P ≤ .046). PTEN loss (58%) was detected in type I endometrial carcinoma of grade 1, at early stage, with mismatch repair gene loss (24.4%) and microsatellite instability-positive status (22%; P ≤ .05). Nuclear ß-catenin (16%) was found in type I tumors of younger patients (P ≤ .003). In contrast, BRAF-V600E mutations were not detected (0%). Mammalian target of rapamycin cytoplasmic high expression implied poorer prognosis (P = .02; Kaplan-Meier, log-rank test), but grade 3 tumors, vascular invasion, advanced stage, or PTEN presence correlated independently with a negative impact on survival (all P ≤ .036; Cox analysis). Our results show that mammalian target of rapamycin, PTEN, and ß-catenin are independently involved in different molecular subtypes of endometrial carcinoma with diverse patients' prognosis and support their distinctive treatment based on targeted drugs.