Marina Chekmareva

Localization of prostate cancer metastasis-suppressor activity on human chromosome 17

Prostate cancer is the most commonly diagnosed malignancy in American men. Currently, it is difficult to accurately predict the clinical course of histologically localized prostatic cancer in the individual patient. Identification of markers for metastatic potential of prostate cancer may improve the diagnosis and treatment of this disease. We have previously demonstrated that human chromosome 17 (17pter–q23) suppresses the metastatic ability of AT6.1 rat prostatic cancer cells. In this study we report on the further localization of the metastasis suppressor activity encoded by human chromosome 17.

Identification of Function for CD44 Intracytoplasmic Domain (CD44-ICD) MODULATION OF MATRIX METALLOPROTEINASE 9 (MMP-9) TRANSCRIPTION VIA NOVEL PROMOTER RESPONSE ELEMENT

Background: CD44, a multifunctional receptor, undergoes cleavage to produce an intracytoplasmic domain (CD44-ICD) that translocates into the nucleus. Results: CD44-ICD binds to a novel DNA consensus sequence and activates many genes. Conclusion: We finally explain the multifunctionality of CD44 and reveal new genes affected by CD44. Significance: Our findings will accelerate the understanding of how CD44-ICD regulates a multitude of cell functions. CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.

Immunohistochemical Differences Between Mucinous and Microglandular Adenocarcinomas of the Endometrium and Benign Endocervical Epithelium

Mucinous and microglandular adenocarcinomas of the endometrium (MUC-AD and MIGL-AD, respectively) are uncommon types of endometrial cancer. When present in endometrial biopsy or curettage, these tumors may display a unique microglandular architectural pattern mimicking benign microglandular hyperplasia (MGH) of the endocervix. We compared the immunoprofile of MUC-AD and MIGL-AD with that of MGH and benign endocervical glands to identify the markers that would reliably separate these malignancies from benign endocervical tissue. A total of 10 MIGL-AD and 30 MUC-AD cases were collected for the study. Fifteen consecutive cases of benign endocervical glands and MGH were used as a control group. All cases were stained for vimentin, p16, Ki-67, BCL-2, survivin, CD10, and CD34. p16 was the only marker that showed a significantly different staining pattern between the benign and malignant cases, whereas the staining for vimentin, Ki-67, BCL-2, and survivin demonstrated marked overlaps. All but 1 MUC-AD and MIGL-AD cases were positive for p16, whereas none of the cases of benign mucinous endocervical epithelium and MGH showed p16 positivity. Furthermore, the stromal cells of endocervix demonstrated weak to moderate positivity for CD10 and strong positivity for CD34, whereas endometrial tumors showed a reverse pattern, with strong stromal positivity for CD10 and either no, or only weak, staining for CD34. In conclusion, epithelial p16 and stromal CD10/CD34 immunostaining can be useful in distinguishing MUC-AD and MIGL-AD from benign endocervical epithelium in endometrial sampling.

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion.

In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.

Does the presence of adenomyosis and lymphovascular space invasion affect lymph node status in patients with endometrioid adenocarcinoma of the endometrium

OBJECTIVE We sought to determine the prevalence of adenomyosis and assess its effect on lymph node status in endometrioid adenocarcinoma of the endometrium (EAC). STUDY DESIGN Hysterectomy specimens from a single institution were reviewed for the presence of adenomyosis, lymphovascular space invasion (LVSI), tumor grade, histology, and lymph node status. Standard statistical analysis was used to compare variables. RESULTS Adenomyosis was present in 42% of total and 66% of malignant hysterectomy specimens (P = .009). Adenomyosis was most commonly associated with EAC histology (P = .023). LVSI was found to be an independent predictor of lymph node metastasis in EAC patients without adenomyosis, but not in those with coexisting adenomyosis (odds ratio, 58.7; P = .03; and odds ratio, 4.98; P = .15; respectively). CONCLUSION Adenomyosis was associated with a lower risk of lymph node metastasis in EAC patients with LVSI. Further studies are needed to investigate the role of adenomyosis in lymphatic tumor infiltration.

Accurate Characterization of Benign and Cancerous Breast Tissues: Aspecific Patient Studies using Piezoresistive Microcantilevers

Breast cancer is the largest detected cancer amongst women in the US. In this work, our team reports on the development of piezoresistive microcantilevers (PMCs) to investigate their potential use in the accurate detection and characterization of benign and diseased breast tissues by performing indentations on the micro-scale tissue specimens. The PMCs used in these experiments have been fabricated using laboratory-made silicon-on-insulator (SOI) substrate, which significantly reduces the fabrication costs. The PMCs are 260 μm long, 35 μm wide and 2 μm thick with resistivity of order 1.316×10(-3) Ω cm obtained by using boron diffusion technique. For indenting the tissue, we utilized 8 μm thick cylindrical SU-8 tip. The PMC was calibrated against a known AFM probe. Breast tissue cores from seven different specimens were indented using PMC to identify benign and cancerous tissue cores. Furthermore, field emission scanning electron microscopy (FE-SEM) of benign and cancerous specimens showed marked differences in the tissue morphology, which further validates our observed experimental data with the PMCs. While these patient aspecific feasibility studies clearly demonstrate the ability to discriminate between benign and cancerous breast tissues, further investigation is necessary to perform automated mechano-phenotyping (classification) of breast cancer: from onset to disease progression.

ERG and CHD1 heterogeneity in prostate cancer: use of confocal microscopy in assessment of microscopic foci.

Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core.

The Role of Motility Proteins and Metastasis‐Suppressor Genes in Prostate Cancer Progression

In 1996, an estimated 317,000 new cases of prostate cancer will be diagnosed in the United States. The incidence of prostate cancer has more than doubled in the past five years; in fact, it is estimated that aggressive screening starting at age 50 could potentially identify 10,000,000 American men with histologically localized prostate cancer. In order to reduce deaths from prostate cancer, it is necessary not only to diagnose but also to accurately predict the clinical course of an individual patients cancer, thus allowing for more effectively directed treatment.

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma.

Carbonic anhydrase 4 and crystallin alpha-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology

BACKGROUND Thyroid nodules are present in 19%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules. METHODS A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone. RESULTS Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99. CONCLUSIONS Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.