Cancer stem cells have been defined as cells within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Experimental evidence showed that these highly tumorigenic cells might be responsible for initiation and progression of cancer into invasive and metastatic disease. Eradicating prostate cancer stem cells, the root of the problem, has been considered as a promising target in prostate cancer treatment to improve the prognosis for patients with advanced stages of the disease.

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Prostate Cancer Stem Cells: Research Advances.

Cluster of differentiation 44 (CD44), a well-known transmembrane glycoprotein, serves as a promoting factor in the carcinogenesis and progression of a variety of neoplasms. Previous studies have demonstrated that aberrant expression of CD44 was associated with the initiation, invasion, metastasis, and therapy-resistance of breast cancer, but whether there was any association between CD44 and pathological characteristics of breast cancer or epidermal growth factor receptor (EGFR) has not been clearly elucidated. In this study, we utilized public microarray data analysis and tissue microarray technologies to display that CD44 level was enhanced in breast cancer and was significantly correlated with histological grade and the status of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 (HER2) and EGFR. Furthermore, mRNA expression of CD44 in breast tumors was positively correlated with basal cytokeratin markers KRT5 and KRT17, but inversely associated with luminal marker FOXA1. Besides, Kaplan-Meier analysis showed that high CD44 mRNA level had adverse impact on the progression-free survival of patients with HER2-expressing or basal-like breast cancer. Functionally, inhibition of EGFR activity by erlotinib impaired the invasion and migration ability of breast cancer cell lines. Western blot assays demonstrated that erlotinib treatment decreased the expression of CD44, accompanied with the reduced protein levels of mesenchymal and cancer stem cell markers. Collectively, this study suggested that the expression of CD44 was upregulated by EGFR pathway and CD44 had a robust impact on the development of breast cancer.

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CD44 correlates with clinicopathological characteristics and is upregulated by EGFR in breast cancer

Aim To determine frequency of tumors with immunohistochemical markers of cancer stem cells (CSC) CD44+/CD24- in patients with breast cancer (BC) of different molecular subtype and to evaluate their prognostic value. Object Surgical material of 132 patients with BC stage I-II, age from 23 to 75 years, mean age - 50.2 ± 3.1 years was studied. Methods Clinical, immunohistochemical (expression CD44+/CD24-), morphological, statistical. Results BC is characterized by heterogeneity of molecular subtypes and expression of markers (CD44+/CD24-). Immunohistochemical study of expression of CSC markers in surgical material has detected their expression in 34 (25.4%) patients with BC of different molecular subtypes. The highest frequency of cells with expression of CSC marker was observed in patients with basal molecular subtype (44.8% patients). Most of BC patients with phenotype CD44+/CD24 had stage I of tumor process (34.3%). Statistical processing of data has showen that Yule colligation coefficient equaled 0.28 (р > 0.05) that argues poor correlation between stage of tumor process and number of tumors with positive expression of CSC markers. Statistical processing of data has showen high correlation between presence of cells with expression of CSC markers and metastases of BC in regional lymph nodes (Yule colligation coefficient equals 0.943; р 0.05). Conclusion Significance of tumor cells with markers CD44+/CD24- within the limits of molecular subtype of BC may be additional criterion for advanced biological characteristic of BC, and in patients with BC of basal molecular subtype - for predictive evaluation of individual potential of tumor to aggressive clinical course.

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СD44+/CD24- markers of cancer stem cells in patients with breast cancer of different molecular subtypes.

The hypermethylation of estrogen receptor alpha (ERα) promoter is a common molecular alteration in sporadic breast cancer (BC), but its involvement in familial BC remains largely unknown. In the present study, we analyzed the methylation statuses of four regions (ER1, ER3, ER4, and ER5) of the ERα promoter and the ERα expression levels of 113 familial BC patients in a Han Chinese Population from northeastern China and evaluated the association between major clinicopathological features and the hypermethylation statuses of the ERα gene. Tumor samples were analyzed for ERα methylation status by the methylation-specific polymerase chain reaction for ERα, PR, p53, BRCA-1, and BRCA-2 by immunohistochemical (IHC) staining and for Her-2 status by IHC and fluorescence in situ hybridization (FISH). ERα methylation was observed in tumor tissues in 47/113 (41.6%) familial BC patients. There were no significant differences in the methylation statuses among ER1 (20.4%), ER3 (18.6%), ER4 (17.7%), and ER5 (19.5%; χ2 = 3.89, p > 0.05). An association between ERα expression level and its promoter methylation level was found. In addition, ERα methylation was significantly correlated with tumor size, PR expression, p53 nuclear accumulation, and BRCA-1 and BRCA-2 statuses. In conclusion, in familial BC patients, the level of ERα gene promoter methylation correlates with ERα expression, PR, p53 nuclear accumulation, and BRCA-1 and BRCA-2 statuses. Epigenetic alteration of ERα gene may play an important role in the pathogenesis of familial BC.

Promoter methylation status and expression of estrogen receptor alpha in familial breast cancer patients

Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH)+ and CD44+CD24-] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH+ or CD44+CD24- subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.

A preliminary study of the role of extracellular -5'- nucleotidase in breast cancer stem cells and epithelial-mesenchymal transition.

Immunohistochemically detected BRCA1 positivity has been reported to indicate the presence of full-length functional protein, while immunohistochemical negativity may be a result of sporadic BRCA1 mutation. We set out to study the immunohistochemical expression of BRCA1 protein in breast cancer and assess associations with lymph node status, histologic grade and expression of other biomarkers. We examined 100 patients aged from 21 to 71 years diagnosed with invasive ductal carcinoma. Immunohistochemistry was performed using anti-BRCA1, ER, PR, HER2/neu, and Ki-67 (MIB-1) antibodies. Cytoplasmic or nuclear-cytoplasmic BRCA1 expression was identified in a total of 64 breast cancer patients. None of the breast cancer tissue samples showed solely nuclear BRCA1 immunoreactivity. BRCA1 expression was associated with higher histologic grade, and majority of BRCA1-positive patients were below 50 years of age. Most BRCA1-negative patients had intermediate grade tumors. BRCA1 expression was positively associated with ER and PR positivity, and negatively associated with HER2/neu overexpression. Although immunohistochemistry can be an inexpensive and valuable preliminary method for detecting the BRCA1 status, BRCA1 protein localization is a complex issue. Well designed studies are needed to further investigate the performance of various anti-BRCA1 antibodies in formalin- fixed paraffin-embedded tissue samples, assess their association with BRCA1 gene mutations and determine clinical usefulness of BRCA1 immunohistochemistry.

Immunohistochemical expression of BRCA1 protein in invasive ductal carcinoma of the breast.

Breast cancer is the most widespread pathology among women. Despite the current progresses in research and treatment of metastatic breast cancer, mortality caused by this disease is still high, because above mentioned therapy is limited due to existence of cells resistant to therapy . Cancer stem cells are the only cells with ability of unlimited proliferative activity and cancerous potential, thus, they participate in the growth, progression and dissemination of cancer. Cancer stem cells are resistant to various forms of therapy, including chemotherapy and radiotherapy . Results of examination showed that 50% of all cases are positive on so called markers of stem cells, thus 45% of cases are negative. CD44+/CD24-low cases (cases that reveal stem cell-phenotype) in the group of invasive ductal carcinoma of Luminal A molecular subtype are almost as many as CD44+/CD24+ and CD44-/CD24+ phenotype cancers. In this group non-stem phenotype cases are 65%, so 5 times more than stem cell phenotype cancers. 1324 postoperative breast materials studied through 2008-2012 at the laboratory of "Pathgeo-Union of Pathologists" LTD and Academician N. Kipshidze Central University Clinic were used as test materials and specimens from 393 patients with invasive ductal carcinoma were selected. CD44/CD24 markers' expression in phenotypically different cancers and clinic-pathologic parameters as well as various biological features was conducted by the Pearson's correlation analysis and using X2 test. Statistical analysis of obtained numeral data was held using SPSS V.19.0 program. Confidence interval of 95% was considered statistically significant. Stem cell phenotype positive cases are with the highest percentage represented in Luminal B and basal-like molecular subgroup that to our minds is associated with their aggressive behavior and resistance to chemotherapy. Relatively good prognosis and response to chemotherapy of Luminal A molecular subtype cancers are to be stipulated by lower percentage of cases with stem cells phenotype. With regard to the dimension of cancer the analysis of stem cell phenotype cancers showed that frequency of stem cell phenotype (CD44+/CD24-low) dramatically increases from T1 to T4 cancers. High density of stem cell phenotype cancers in cancers with metastatic lymphatic nodes proves that presence of mentioned phenotype plays a role in progression and dissemination. On the one hand, little amount of stem cells phenotype cancers (CD44+/CD24-low), on the other hand absence of negative cases for markers of stem cell in Her2 subtype makes us consider that come phenotype, close to stem-cell phenotype, plays the leading role in Her2 positive cases.

Features of CD44+/CD24-low phenotypic cell distribution in relation to predictive markers and molecular subtypes of invasive ductal carcinoma of the breast.

Despite advances in detection and treatment of breast cancer, mortality from this disease remains high. According to contemporary point of view the reason for this lies in fact, that in addition to intertumor heterogeneity, there is also a high degree of intratumor diversity in cancer cell population. For most cancers it is less clear which cells within the tumor clone possess tumor-initiating cell function. During studying oncogenesis and maligniяation processes a pool of cancer cells with stem characteristics - cancer stem cells (CSC) was identified. Indeed, the specifications of them let us conclude, that exactly these cells comprise the leading substrate for cancer initiation and self-renewal. Breast carcinomas have been reported to contain a subpopulation of CD44(+)/CD24(-)/low cancer cells, which are capable of generating tumors even when implanted in very low numbers. Exactly these cells are considered to be CSCs in different subtypes of breast cancer and cancers with CD44(+)/CD24(-)/low phenotype are confirmed to have a poor prognosis (but some controversies remain concerning this issue). The aim of the review was to assess the current literature published on the breast cancer stem cells. There are not so many studies, revealing the diversity of cancer stem cells in different types, different sizes and different grade of breast cancers. CSC distribution in breast cancer with lymph node involvement, metastasizing and chemoresistant cases, existence of circulating tumor cells in not studied precisely. So the concept of cancer stem cells in breast cancer is still a topic for discussions. This can bring light to a better understanding of the pathological process in breast cancer and ways to target it.

Distribution of cancer stem cells in ductal invasive carcinoma of breast (review).

The purpose of our study was to learn the distribution characteristics of cancer stem cell markers (CD24, CD44) in invasive carcinomas with different grade and molecular subtype. For research was used 1324 postoperative breast cancer samples, from which were selected 393 patient with invasive ductal carcinoma samples examined 2008-2012 in Laboratory of "Pathgeo Union of Pathologist" is and N.Kipshidze Central University Hospital. The age range is between 23-73 year. For all cases were performed immunohistochemical study using ER, PR, Her2, Ki67, CK5- molecular markers (Leica Microsystems). For identify cancer stem cells mononuclear antibodies CD24 (BIOCARE MEDICAL, CD44 - Clone 156-3C11; CD24 - Clone SN3b) were used. Association of CD44/CD24 expression in different subtypes of cells, between clinicopathological parameters and different biological characteristics were performed by Pearson correlation and usind X2 tests. Obtained quantitative statistical analyses were performed by using SPSS V.19.0 program. Statistically significant were considered 95% of confidence interval. The data shows, that towards G1-G3, amount of CD44 positive cases increased twice. CD44 positive cases are evenly distributed between Luminal A, Luminal B, HER2+, triple negative basal like cell subtypes and in significantly less (4,8 times) in Her2+ cases. Maximum amount of CD44 negative cases is shown in Luminal A subtype, which could be possible cause of better prognosis and high sensitivity for chemotherapy. For one's part such aggressive subtypes of breast cancer as Luminal B and basal like cell type, are characterized by CD44 positive and antigen high expression, which can be reason of aggressive nature of this types and also reason of chemotherapy resistance. As well as amount of CD24 positive cases according to malignancy degree, also antigen expression features does not show any type of correlation between malignancy degree and CD24 positivity or with CD24 expression features, or presence of stem cells. That can be the reason of tumor aggressivity and chemoresistance. exceptions are Her2 positive tumors because they have different base of carcinogenesis.

M Gudadze

Papers

Immunohistochemical expression of BRCA1 protein in invasive ductal carcinoma of the breast.

Features of CD44+/CD24-low phenotypic cell distribution in relation to predictive markers and molecular subtypes of invasive ductal carcinoma of the breast.

Distribution of cancer stem cells in ductal invasive carcinoma of breast (review).

Distribution of CD44/CD24 positive cells in ductal invasive carcinoma of breast of different grade and molecular subtype.