Estrogens Correlate with PELP1 Expression in ER Positive Breast Cancer

The Proline-, glutamic acid- and leucine-rich protein 1 (PELP1) is an estrogen receptor (ER) coactivator and a proto-oncogene known to be deregulated in endocrine cancers. In breast cancer, PELP1 overexpression has been associated with endocrine therapy resistance. Although PELP1 is known to be regulated by estrogens in vitro, its association with estrogen levels within the tissue of breast cancer patients has not previously been assessed. Here, we determined PELP1 mRNA expression levels in paired samples of normal and malignant breast tissue obtained from 32 postmenopausal and 11 premenopausal women. In the total sample set, PELP1 levels were higher in tumors compared to normal breast tissue (P = 0.041). Among postmenopausal women, PELP1 tumor levels correlated positively with estrone (E₁) and estradiol (E₂) levels in both normal tissue (r = 0.543, P = 0.003 and r = 0.601, P = 0.001, respectively) and plasma (r = 0.392, P = 0.053 and r = 0.403, P = 0.046, respectively). Analyzing all ER+ tumors (n = 26), PELP1 correlated positively with E₁ and E₂ in tumor tissue (r = 0.562, P = 0.003 and r = 0.411, P = 0.037, respectively) and normal tissue (r = 0.461, P = 0.018 and r = 0.427, P = 0.030, respectively) in addition to plasma E₁, E₂ and estrone sulphate (E₁S) concentrations (r = 0.576, P = 0.003, r = 0.456, P = 0.025 and r = 0.406, P = 0.049, respectively). Finally, PELP1 correlated positively with ER mRNA (ESR1) (r = 0.553, P = 0.026) in ER+ tumors, whereas a negative association between PELP1 and ESR1 (r = -0.733, P = 0.010) was observed in ER- breast tumors. Taken together, tumor PELP1 mRNA expression is associated with estrogen levels in breast cancer, suggesting a potentially important role of PELP1 in ER+ breast cancer growth in vivo.     

SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization

Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.     

Estrogen Receptor and HER2 Status on Disseminated Tumor Cells and Primary Tumor in Patients with Early Breast Cancer

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.     

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.     

Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer

Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.     

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.     

RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.     

Tumor Infiltrating Lymphocytes and NHERF1 Impact on Prognosis of Breast Cancer Patients

BACKGROUND: Breast cancer (BC) is a heterogeneous disease, and patients with apparently similar clinicopathological characteristics in clinical practice show different outcome. This study evaluated in primary BCs and in the subgroup of the triple-negative breast cancers (TNBCs) the level of tumor infiltrating lymphocytes (TILs), Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression, and their association respect to the clinical outcome of patients. MATERIAL AND METHODS: NHERF1 expression was assessed by immunohistochemistry in 338 BC samples; the analysis of TILs was examined using hematoxylin and eosin stained slides, according to International TILs Working Group 2014. RESULTS: Multivariate analysis identified TILs as an independent prognostic factor for DFS in the entire cohort and in the TNBC subgroup (HR, 0.32; 95% CI, 0.12–0.87; P = 0.026; and HR, 0.22; 95% CI, 0.06–0.80; P = 0.022, respectively). Univariate and survival analysis by Kaplan–Meier method revealed that patients with cytoplasmic (c) NHERF1^-/TILs⁺ expression had better DFS than other patients (P = 0.049), and this result was also found in the TNBC subgroup (P = 0.031). Moreover, TNBC patients with cNHERF1⁻/TILs⁻ expression had a worse DFS and OS than other patients (P = 0.057 and P = 0.002, respectively). CONCLUSIONS: In the complex scenario of BC and in the era of tumor immunogenicity and immunotherapy, we found an association of TIL levels and cNHERF1 expression that could be useful to identify BCs and particularly TNBC patients with different prognosis and clinical outcome.     

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14⁺HLA-DR^low/-CD86^low/-CD80^low/-CD163^low/-) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.     

CoREST1 Promotes Tumor Formation and Tumor Stroma Interactions in a Mouse Model of Breast Cancer

Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.     

Upregulation of SATB1 Is Associated with Prostate Cancer Aggressiveness and Disease Progression

Disease aggressiveness remains a critical factor to the progression of prostate cancer. Transformation of epithelial cells to mesenchymal lineage, associated with the loss of E-cadherin, offers significant invasive potential and migration capability. Recently, Special AT-rich binding protein (SATB1) has been linked to tumor progression. SATB1 is a cell-type restricted nuclear protein, which functions as a tissue-specific organizer of DNA sequences during cellular differentiation. Our results demonstrate that SATB1 plays significant role in prostate tumor invasion and migration and its nuclear localization correlates with disease aggressiveness. Clinical specimen analysis showed that SATB1 was predominantly expressed in the nucleus of high-grade tumors compared to low-grade tumor and benign tissue. A progressive increase in the nuclear levels of SATB1 was observed in cancer tissues compared to benign specimens. Similarly, SATB1 protein levels were higher in a number of prostate cancer cells viz. HPV-CA-10, DU145, DUPro, PC-3, PC-3M, LNCaP and C4-2B, compared to non-tumorigenic PZ-HPV-7 cells. Nuclear expression of SATB1 was higher in biologically aggressive subclones of prostate cancer cells with their respective parental cell lines. Furthermore, ectopic SATB1 transfection conferred increased cell motility and invasiveness in immortalized human prostate epithelial PZ-HPV-7 cells which correlated with the loss of E-cadherin expression. Consequently, knockdown of SATB1 in highly aggressive human prostate cancer PC-3M cells inhibited invasiveness and tumor growth in vivo along with increase in E-cadherin protein expression. Our findings demonstrate that SATB1 has ability to promote prostate cancer aggressiveness through epithelial-mesenchymal transition.     

乳腺癌患者循环肿瘤细胞与癌干细胞相关性的初步研究

目的：检测乳腺癌患者外周血单核细胞（PBMC）中循环肿瘤细胞（CTC）和具有癌干细胞（CSC）标志的CTC（CSC-CTC）,探讨患者外周血微转移与CSC的相关性。方法：患者和健康者PBMC与磁珠偶联上皮细胞黏附分子单抗孵育后,用磁性分离法富集PBMC中的上皮细胞。以CK＋为患者PBMC中CTC标志,用流式细胞术（FCM）检测健康者和患者的PBMC中CK＋细胞及CK＋/CD44＋/CD24-细胞含量,并比较各组间CTC、CSC-CTC含量的差异。结果：用FCM在73.07%的患者中检测到CTC,在19例检测到CTC的患者中18例有CSC-CTC（94.74%）,CTC中CSC数量比例平均为19.01%,且患者PBMC中CTC和CSC-CTC比例与临床TNM分期相关。结论：初步建立了患者外周血CSC-CTC的检测方法,结果显示乳腺癌患者外周血微转移中有CSC-CTC的参与,临床分期越晚的患者CTC和CSC-CTC的数量越多。     

趋化因子受体CXCR1、CXCR2与肿瘤研究进展

趋化因子受体是由7个跨膜区组成的G蛋白偶联受体,多个系统的肿瘤细胞均表达趋化因子受体,其在肿瘤的发生、发展和转移等各个阶段都发挥重要作用.近年来有不少研究发现趋化因子受体中的CXCR1和CXCR2与肿瘤关系密切,认为其可能成为肿瘤治疗的一个潜在新靶点.本文就CXCR1和CXCR2这两种趋化因子受体与肿瘤的关系做一综述.