Epigenetic and Pluripotency Aspects of Disseminated Cancer Cells During Minimal Residual Disease.

Our understanding of the minimal residual disease (MRD) in solid cancers indicates that it can persist in the system for years or even decades. We now know that the persistence of MRD might depend on the dormancy of the disseminated cancer cells (DCCs). Once DCCs exit dormancy, they become metastatic and the survival rates of the patients inevitably decrease. Thus, innovative treatments are required to extend the asymptomatic phase of MRD after the initial therapeutic intervention. With the latest advances in cancer research, there is a greater need to explore and understand the biology, timing of dissemination, and origin of DCCs during tumor progression. These important aspects of DCCs impact the selection, design, administration, and timing of effective therapies. Herein, we summarize the current understanding of MRD biology in solid tumors, with a focus on epigenetics and pluripotency, presenting an overall view of the direction the field is taking to reach the goal of reducing cancer-related mortalities that result from metastasis.

Gene expression profile and cancer-associated pathways linked to progesterone receptor isoform a (PRA) predominance in transgenic mouse mammary glands.

BACKGROUND: Progesterone receptor (PR) is expressed from a single gene as two isoforms, PRA and PRB. In normal breast human tissue, PRA and PRB are expressed in equimolar ratios, but isoform ratio is altered during malignant progression, usually leading to high PRA:PRB ratios. We took advantage of a transgenic mouse model where PRA isoform is predominant (PRA transgenics) and identified the key transcriptional events and associated pathways underlying the preneoplastic phenotype in mammary glands of PRA transgenics as compared with normal wild-type littermates. METHODS: The transcriptomic profiles of PRA transgenics and wild-type mammary glands were generated using microarray technology. We identified differentially expressed genes and analyzed clustering, gene ontology (GO), gene set enrichment analysis (GSEA), and pathway profiles. We also performed comparisons with publicly available gene expression data sets of human breast cancer. RESULTS: We identified a large number of differentially expressed genes which were mainly associated with metabolic pathways for the PRA transgenics phenotype while inflammation- related pathways were negatively correlated. Further, we determined a significant overlap of the pathways characterizing PRA transgenics and those in breast cancer subtypes Luminal A and Luminal B and identified novel putative biomarkers, such as PDHB and LAMB3. CONCLUSION: The transcriptional targets identified in this study should facilitate the formulation or refinement of useful molecular descriptors for diagnosis, prognosis, and therapy of breast cancer.

PHLDA1 (pleckstrin homology-like domain, family A, member 1) knockdown promotes migration and invasion of MCF10A breast epithelial cells.

PHLDA1 (pleckstrin homology-like domain, family A, member 1) is a multifunctional protein that plays distinct roles in several biological processes including cell death and therefore its altered expression has been identified in different types of cancer. Progressively loss of PHLDA1 was found in primary and metastatic melanoma while its overexpression was reported in intestinal and pancreatic tumors. Previous work from our group showed that negative expression of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer disease. However, the function of PHLDA1 in mammary epithelial cells and the tumorigenic process of the breast is unclear. To dissect PHLDA1 role in human breast epithelial cells, we generated a clone of MCF10A cells with stable knockdown of PHLDA1 and performed functional studies. To achieve reduced PHLDA1 expression we used shRNA plasmid transfection and then changes in cell morphology and biological behavior were assessed. We found that PHLDA1 downregulation induced marked morphological alterations in MCF10A cells, such as changes in cell-to-cell adhesion pattern and cytoskeleton reorganization. Regarding cell behavior, MCF10A cells with reduced expression of PHLDA1 showed higher proliferative rate and migration ability in comparison with control cells. We also found that MCF10A cells with PHLDA1 knockdown acquired invasive properties, as evaluated by transwell Matrigel invasion assay and showed enhanced colony-forming ability and irregular growth in low attachment condition. Altogether, our results indicate that PHLDA1 downregulation in MCF10A cells leads to morphological changes and a more aggressive behavior.

Clinical relevance of galectin-1 expression in non-small cell lung cancer patients.

BACKGROUND: Identification of biomarkers in lung cancer, a leading cause of cancer-related mortality, has a meaningful clinical relevance in the quest of novel prognostic factors and therapeutic targets. The glycan-binding protein galectin-1 (Gal-1) modulates tumor progression by mediating cell-cell and cell-extracellular matrix interactions, as well as angiogenesis and tumor immune-escape. Previous works reported the expression of Gal-1 in lung cancer, although its clinical significance remains uncertain. OBJECTIVE: To assess the clinicopathologic relevance and prognostic value of Gal-1 expression in a cohort of 103 Stage I-III non-small cell lung cancer (NSCLC) patients. METHODS: Gal-1 expression was determined by immunohistochemistry in tumor tissue samples. The percentage of immunoreactive tumor cells and stroma, as well as the presence of blood vessels with positively stained endothelium in the tumor and surrounding normal tissue, were recorded. Results were correlated with the clinicopathologic factors of the patients (Spearman's rank correlation coefficient, chi-square test) and overall survival by univariate (Kaplan Meier) and multivariate analyses (Cox regression hazard model). RESULTS: We did not observe significant associations between Gal-1 expression and relevant clinicopathologic features at diagnosis of NSCLC. However, Kaplan Meier analysis revealed a significant association between Gal-1 expression and overall survival, when Gal-1 expression was analyzed on tumor cells alone ("tumor cell percentage") or when an integrated score accounting for tumor cell as well as stromal expression of Gal-1 ("total score") was assessed. Patients showing high Gal-1 expression evidenced a poorer clinical outcome. Furthermore, "total score" remained significantly associated with survival by multivariate Cox regression analysis in the whole cohort of patients, even when controlling for the classical predictors and prognostic factors of NSCLC. CONCLUSION: We conclude that Gal-1 expression may be a useful biomarker for better prediction of the clinical outcome and management of NSCLC patients.

TGF-beta specifically enhances the metastatic attributes of murine lung adenocarcinoma: implications for human non-small cell lung cancer.

Lung cancer is the most frequent and one of the most deadly cancer types and is classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Transforming growth factor beta (TGFß) regulates a wide array of cell functions and plays a major role in lung diseases, including NSCLC. TGFß signals through the complex of TGFß type I and type II receptors, triggering Smad and non-Smad signaling pathways such as PI3K/Akt and MEK1/ERK. We investigated the role of TGFß1 on the progression of the murine lung adenocarcinoma cell line LP07. Furthermore, we undertook a retrospective study with tissue samples from stage I and II NSCLC patients to assess the clinical pathologic role and prognostic significance of TßRI expression. We demonstrated that although lung cancer cell monolayers responded to TGFß1 anti-mitogenic effects and TGFß1 pulse (24 h treatment) delayed tumor growth at primary site; a switch towards malignant progression upon TGFß1 treatment was observed at the metastatic site. In our model, TGFß1 modulated in vitro clonogenicity, protected against stress-induced apoptosis and increased adhesion, spreading, lung retention and metastatic outgrowth. PI3K and MEK1 signaling pathways were involved in TGFß1-mediated metastasis stimulation. Several of these TGFß responses were also observed in human NSCLC cell lines. In addition, we found that a higher expression of TßRI in human lung tumors is associated with poor patient's overall survival by univariate analysis, while multivariate analysis did not reach statistical significance. Although additional detailed analysis of the endogenous signaling in vivo and in vitro is needed, these studies may provide novel molecular targets for the treatment of lung cancer.

Estrogen receptor ß and epidermal growth factor receptor as early-stage prognostic biomarkers of non-small cell lung cancer.

As 20% of stage I NSCLC patients develop recurrent and often incurable cancer, the identification of prognostic markers has a meaningful clinical application. The biological significance of steroid hormone and EGF receptors, able to regulate key physiological functions, remains elusive in NSCLC. Our aim was to investigate the prognostic input of estrogen receptors (ERα, ERß), progesterone receptors (PR) and EGFR in tumors from 58 stage I NSCLC patients. Antigen expression was analyzed by immunohistochemistry. Prognostic evaluation was performed with the multivariate Cox model. We found that about 70 and 40% of samples expressed ERα or ERß at cytoplasmic or nuclear level, respectively. Besides, only 12.1% of samples weakly expressed nuclear PR and 62.7% showed membrane EGFR staining. Correlation studies indicated an inverse association between EGFR expression and smoking status (p<0.01). Multivariate studies showed that the lack of nuclear ERß or the loss of EGFR expression were independent prognosis markers associated with shorter overall survival. We also found that patients whose tumors were negative for these two biomarkers presented the worst outcome. In conclusion, our findings could be useful for selecting stage I NSCLC patients with poor prognosis to apply an earlier treatment that impacts on survival.

Mast cell phenotypes and microvessels in non-small cell lung cancer and its prognostic significance.

The impact of interstitial inflammatory cells, such as mast cells, and angiogenesis on the prognosis of cancer patients has been reported in many solid tumors, although there is disagreement about their role. We undertook a retrospective study with tissue samples from 65 patients with stage I and II non-small cell lung cancer to assess the clinical pathologic role and prognostic significance of mast cells. Mast cell phenotypes were identified by immunohistochemistry for tryptase and chymase. In addition, we identified microvessels using the endothelial marker CD34. Mast cell and microvessel density was quantified by assessing immunopositive cells in the intratumoral and peritumoral zones of tumor specimens. Both mast cell and microvessel density was higher in the peritumoral zone than the intratumoral zone (P