MiR-185 is involved in human breast carcinogenesis by targeting Vegfa

MiR-185 expression has been associated with many cancers. However, the roles of miR-185 in human breast cancer remain elusive. Here, we found that miR-185 expression was decreased in human breast cancer tissues compared with healthy tissue controls. Up-regulation of miR-185 inhibited breast cancer cell proliferation and invasion and vice versa. MiR-185 was shown to bind to the 3′-untranslated region (UTR) of vascular endothelial growth factor a (Vegfa), and a significant inverse correlation was found between miR-185 and Vegfa. Vegfa overexpression partially restored the inhibition of cell proliferation and invasion that was induced by miR-185, and vice versa. Additionally, Vegfa expression was found to be high in human breast cancer tissues. Thus, miR-185-mediated Vegfa targeting may be involved in breast cancer formation.     

Epigenetic regulation of glycosylation and the impact on chemo-resistance in breast and ovarian cancer

Glycosylation is one of the most fundamental posttranslational modifications in cellular biology and has been shown to be epigenetically regulated. Understanding this process is important as epigenetic therapies such as those using DNA methyltransferase inhibitors are undergoing clinical trials for the treatment of ovarian and breast cancer. Previous work has demonstrated that altered glycosylation patterns are associated with aggressive disease in women presenting with breast and ovarian cancer. Moreover, the tumor microenvironment of hypoxia results in globally altered DNA methylation and is associated with aggressive cancer phenotypes and chemo-resistance, a feature integral to many cancers. There is sparse knowledge on the impact of these therapies on glycosylation. Moreover, little is known about the efficacy of DNA methyltransferase inhibitors in hypoxic tumors. In this review, we interrogate the impact that hypoxia and epigenetic regulation has on cancer cell glycosylation in relation to resultant tumor cell aggressiveness and chemo-resistance.     

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na⁺-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na⁺. Although taurine uptake was reduced in Cl^- free medium a significant portion of taurine uptake persisted in the presence of NO₃ ^-. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the V_max of influx was increased without affecting the K_m. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na⁺. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na⁺-dependent taurine uptake may not be strictly dependent upon extracellular Cl^-.     

P-glycoprotein down-regulates expression of breast cancer resistance protein in a drug-free state

This study investigated whether P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are linked in terms of expression. RT-PCR and Western blot analyses showed that the lung cancer cell line SK-MES-1/WT expressed BCRP. In a drug-free state, BCRP expression was significantly down-regulated in doxorubicin-resistant SK-MES-1/DX1000 cells overexpressing Pgp. Pharmacological inhibitors (PSC833 or verapamil) or siRNA for Pgp inhibited the down-regulation of BCRP, which was confirmed by confocal microscopy. PSC833 induced the phosphorylation of c-Jun NH2-terminal kinase (JNK) and c-Jun, while the JNK inhibitor SP600125 inhibited this effect. Dominant negative c-Jun decreased the expression of BCRP, but increased that of Pgp. These results indicate that Pgp down-regulates BCRP expression in a drug-free state in which JNK/c-Jun is involved.     

miR-139-5p is a regulator of metastatic pathways in breast cancer

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFβ, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.     

Estrogen receptor β in the breast: role in estrogen responsiveness and development of breast cancer

Breast cancer is one of the most common forms of cancer observed in women. Endogenous estrogen is thought to play a major role in its development and estrogen receptor blockers are the most important drugs in its treatment. It has long been thought that any conditions or exposures, which enhance estrogenic responses, would result in an increased risk for breast cancer. The discovery of the second estrogen receptor, ERβ, which can have effects opposite to those of the well-known ‘original’ estrogen receptor (now called ER) challenges this simplistic view. In order to understand breast cancer one must first understand how the normal breast is maintained. The functions of ERβ in the breast remain to be defined but from what we have learnt about its activities in in vitro systems, this estrogen receptor may have a protective role in the breast. Studies in human and rodent breasts as well as in human breast cancer biopsies reveal that ERβ is by far the more abundant of the two ERs. Despite the role of estrogen in proliferation of the breast, neither of the two ERs appears to located in epithelial cells which divide in response to estrogen. In order to define the functions of ERβ in the normal and malignant breast, we have created mice in which the ERβ gene has been inactivated. Studies of the breasts of ERβ knock out mice (BERKO) revealed abnormal epithelial growth, overexpression of Ki67 and severe cystic breast disease as mice age.     

miR-29a suppresses growth and invasion of gastric cancer cells in vitro by targeting VEGF-A

Increasing data shows miR-29a is a key regulator of oncogenic processes. It is significantly down-regulated in some kind of human tumors and possibly functionally linked to cellular proliferation, survival and migration. However, the mechanism remains unclear. In this study, we report miR-29a is significantly under-expressed in gastric cancer compared to the healthy donor. The microvessel density is negatively related to miR-29a expression in gastric cancer tissues. The ectopic expression of miR-29a significantly inhibits proliferation and invasion of gastric cancer cells. Furthermore, western blot combined with the luciferase reporter assays demonstrate that vascular endothelial growth factor A (VEGF-A) is direct target of miR-29a. This is the first time miR-29a was found to suppress the tumor microvessel density in gastric cancer by targeting VEGF-A. Taken together, these results suggest that miR-29a is a tumor suppressor in gastric cancer. Restoration of miR-29a in gastric cancer may be a promising therapeutic approach. [BMB Reports 2014; 47(1):39-44]     

The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: Implications for cancer therapy and prevention

Background

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.

Methods

MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.

Results

In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion

Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.

General significance

These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.     

Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer

Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.     

Hormonal regulation of tumor suppressor proteins in breast cancer cells

This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB). Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation. The expression of p53 in T47D cells grown for 4–5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups. Supplementation of culture medium containing stripped serum with 0.1–1 nM estradiol (E₂) restored p53 to its level seen in the control within 6–24 h. Under above conditions, treatment of cells with R5020 or RU486 reduced (15–30%) the level of p53. Incubation of cells in E₂-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24–72 h. The E₂-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT). T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments. E₂ and OHT caused P1CAT induction as seen by increased CAT activity: E₂ caused an endogenous increase in the expression of an ICI-sensitive c-Myc form. These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process. Further, E₂ regulates p53 level and pRB activity in a coordinated manner.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.