José Esparza-López

Calcitriol restores antiestrogen responsiveness in estrogen receptor negative breast cancer cells: A potential new therapeutic approach

BackgroundApproximately 30% of breast tumors do not express the estrogen receptor (ER) α, which is necessary for endocrine therapy approaches. Studies are ongoing in order to restore ERα expression in ERα-negative breast cancer. The aim of the present study was to determine if calcitriol induces ERα expression in ER-negative breast cancer cells, thus restoring antiestrogen responses.MethodsCultured cells derived from ERα-negative breast tumors and an ERα-negative breast cancer cell line (SUM-229PE) were treated with calcitriol and ERα expression was assessed by real time PCR and western blots. The ERα functionality was evaluated by prolactin gene expression analysis. In addition, the effects of antiestrogens were assessed by growth assay using the XTT method. Gene expression of cyclin D1 (CCND1), and Ether-à-go-go 1 (EAG1) was also evaluated in cells treated with calcitriol alone or in combination with estradiol or ICI-182,780. Statistical analyses were determined by one-way ANOVA.ResultsCalcitriol was able to induce the expression of a functional ERα in ER-negative breast cancer cells. This effect was mediated through the vitamin D receptor (VDR), since it was abrogated by a VDR antagonist. Interestingly, the calcitriol-induced ERα restored the response to antiestrogens by inhibiting cell proliferation. In addition, calcitriol-treated cells in the presence of ICI-182,780 resulted in a significant reduction of two important cell proliferation regulators CCND1 and EAG1.ConclusionsCalcitriol induced the expression of ERα and restored the response to antiestrogens in ERα-negative breast cancer cells. The combined treatment with calcitriol and antiestrogens could represent a new therapeutic strategy in ERα-negative breast cancer patients.

MURINE BETAGLYCAN PRIMARY STRUCTURE, EXPRESSION AND GLYCOSAMINOGLYCAN ATTACHMENT SITES

The primary structure of murine betaglycan, also known as transforming growth factor beta (TGF-beta) type III receptor, was deduced from the nucleotide sequence of a cDNA clone isolated from a heart library. Murine betaglycan is a single spanning membrane polypeptide of 850 amino acids which is highly similar to betaglycan of other species. Transfection of this cDNA into COS1 cells resulted in the expression of a membrane proteoglycan that binds TGF-beta and is recognized by antibodies raised against rat betaglycan. COS1 cells transfected with the double mutant Ser533Ala; Ser544Ala of the murine betaglycan cDNA produced a TGF-beta type III receptor devoid of glycosaminoglycan chains.

Casitas B Lineage Lymphoma b Is a Key Regulator of Peripheral Tolerance in Systemic Lupus Erythematosus

OBJECTIVE To analyze whether the expression and modulation of T cell receptor (TCR) signaling is dependent on Casitas B lineage lymphoma b (Cbl-b) in T cells from patients with systemic lupus erythematosus (SLE) upon stimulation with a tolerogenic substance. METHODS Peripheral blood mononuclear cells were obtained from 20 patients with SLE (active disease or in remission) and 20 healthy controls. Levels of Cbl-b expression were measured using reverse transcription-polymerase chain reaction and Western blotting in peripheral CD4+ T cells from SLE patients and healthy controls upon anergy induction. Cell proliferation was measured using the carboxyfluorescein diacetate succinimidyl ester dilution method. Cytokine production was analyzed by luminometry, and surface expression of activation markers was assessed by flow cytometry. Transfection assays were performed to induce overexpression of Cbl-b, and phosphorylation of TCR-associated kinases was evaluated. RESULTS CD4+ T cells from SLE patients displayed resistance to anergy (as evidenced by increased cell proliferation, interleukin-2 production, and expression of activation and costimulatory markers), and this was associated with altered Cbl-b expression. Upon ionomycin treatment, primary T cells showed enhanced MAPK activity and decreased Akt phosphorylation, which was representative of the anergic state. In T cells from lupus patients, Cbl-b overexpression led to increased expression of phosphorylated MAPK, thus indicating the reversibility of anergy resistance. CONCLUSION These findings suggest that abnormal peripheral tolerance in SLE is caused by a deficiency in Cbl-b, and that this ubiquitin ligase plays a key role in regulating TCR signaling during the induction of peripheral tolerance.

Chronic moderate ethanol intake differentially regulates vitamin D hydroxylases gene expression in kidneys and xenografted breast cancer cells in female mice.

Factors affecting vitamin D metabolism may preclude anti-carcinogenic effects of its active metabolite calcitriol. Chronic ethanol consumption is an etiological factor for breast cancer that affects vitamin D metabolism; however, the mechanisms underlying this causal association have not been fully clarified. Using a murine model, we examined the effects of chronic moderate ethanol intake on tumoral and renal CYP27B1 and CYP24A1 gene expression, the enzymes involved in calcitriol synthesis and inactivation, respectively. Ethanol (5% w/v) was administered to 25-hydroxyvitamin D3-treated or control mice during one month. Afterwards, human breast cancer cells were xenografted and treatments continued another month. Ethanol intake decreased renal Cyp27b1 while increased tumoral CYP24A1 gene expression.Treatment with 25-hydroxyvitamin D3 significantly stimulated CYP27B1 in tumors of non-alcohol-drinking mice, while increased both renal and tumoral CYP24A1. Coadministration of ethanol and 25-hydroxyvitamin D3 reduced in 60% renal 25-hydroxyvitamin D3-dependent Cyp24a1 upregulation (P<0.05). We found 5 folds higher basal Cyp27b1 than Cyp24a1 gene expression in kidneys, whereas this relation was inverted in tumors, showing 5 folds more CYP24A1 than CYP27B1. Tumor expression of the calcitriol target cathelicidin increased only in 25-hydroxyvitamin D3-treated non-ethanol drinking animals (P<0.05). Mean final body weight was higher in 25-hydroxyvitamin D3 treated groups (P<0.001). Overall, these results suggest that moderate ethanol intake decreases renal and tumoral 25-hydroxyvitamin D3 bioconversion into calcitriol, while favors degradation of both vitamin D metabolites in breast cancer cells. The latter may partially explain why alcohol consumption is associated with vitamin D deficiency and increased breast cancer risk and progression.

Primary breast cancer cell culture yields intra-tumor heterogeneous subpopulations expressing exclusive patterns of receptor tyrosine kinases

BackgroundIt has become evident that intra-tumor heterogeneity of breast cancer impact on several biological processes such as proliferation, migration, cell death and also might contribute to chemotherapy resistance. The expression of Receptor Tyrosine Kinases (RTKs) has not been analyzed in the context of intra-tumor heterogeneity in a primary breast cancer cell culture. Several subpopulations were isolated from the MBCDF (M serial-breast cancer ductal F line) primary breast cancer cells and were successfully maintained in culture and divided in two groups according to their morphology and RTKs expression pattern, and correlated with biological processes like proliferation, migration, anchorage-independent cell growth, and resistance to cytotoxic chemotherapy drugs and tyrosine kinase inhibitors (TKIs).MethodsSubpopulations were isolated from MBCDF primary breast cancer cell culture by limiting dilution. RTKs and hormone receptors were examined by Western blot. Proliferation was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT assay). Cell viability was evaluated by Crystal Violet. Migration was assessed using Boyden chambers. Anchorage-independent cell growth was evaluated by colony formation in soft agar.ResultsSeveral subpopulations were isolated from the MBCDF breast cancer cells that were divided into two groups according to their morphology. Analysis of RTKs expression pattern showed that HER1, HER3, c-Met and VEGFR2 were expressed exclusively in cells from group 1, but not in cells from group 2. PDGFR was expressed only in cells from group 2, but not in cells from group 1. HER2, HER4, c-Kit, IGF1-R were expressed in all subpopulations. Biological processes correlated with the RTKs expression pattern. Group 2 subpopulations present the highest rate of cell proliferation, migration and anchorage-independent cell growth. Analysis of susceptibility to chemotherapy drugs and TKIs showed that only Paclitaxel and Imatinib behaved differently between groups. Group 1-cells were resistant to both Paclitaxel and Imatinib.ConclusionsWe demonstrated that subpopulations from MBCDF primary cell culture could be divided into two groups according to their morphology and a RTKs excluding-expression pattern. The differences observed in RTKs expression correlate with the biological characteristics and chemoresistance of each group. These results suggest that intra-tumor heterogeneity contributes to generate groups of subpopulations with a more aggressive phenotype within the tumor.

Interleukin-6 induces epithelial-mesenchymal transition in breast cancer cells

Background: Breast cancer is the leading cause of cancer-related death in Mexico, with most deaths being related to locally advanced or metastatic disease at diagnosis. Epithelial-mesenchymal transition (EMT) is one of the steps that are indispensable for metastasis. Different factors trigger EMT, like TGF-β, EGF and interleukin 6 (IL-6), among others. EMT is characterized by E-cadherin expression loss and N-cadherin and vimentin expression. In this study, we investigated the role of IL-6 on EMT induction. Methods: MBCDF and MBCD17 primary breast cancer cell cultures were used. E-cadherin expression was measured by Western Blot. Cells were stimulated with IL-6 to induce EMT. STAT3 activation was measured using phospho-specific antibodies, and E-cadherin expression was measured as EMT marker. Results: MBCDF and MBCD17 primary breast cancer cell cultures stimulation with IL-6 induced STAT3-Tyr705 phosphorylation without its total levels being altered; in addition, IL-6 cell-stimulation was shown to induce EMT, as evidenced by E-cadherin loss. Conclusions: The results of the present work suggest that IL-6 induces EMT in primary breast cancer cell cultures through STAT3 phosphorylation. (creativecommons.org/licenses/by-nc-nd/4.0/). Si n co nt ar c on e l c on se nt im ie nt o pr ev io p or e sc ri to d el e di to r, no p od rá r ep ro du ci rs e ni f ot oc op ia rs e ni ng un a pa rt e de e st a pu bl ic ac ió n.

Regulation of the Transforming Growth Factor-β Superfamily by Betaglycan

The type III transforming growth factor-β (TGF-β) receptor (TβRIII), also known as betaglycan (BG), is a membrane proteoglycan coreceptor that modulates the action of diverse members of the TGF-β superfamily of growth factors. Membrane bound BG is the precursor of a soluble receptor form generated by the proteolytic cleavage (shedding) of its extracellular region. While membrane BG enhances TGF-β binding to the type II TGF-β receptor, thereby increasing the activity of the factor (1), the soluble form sequesters the ligand, acting as a neutralizing agent of TGF-β (2). Recombinant soluble BG (rSBG) has been used successfully for treatment of diverse experimental pathologies in which TGF-β plays a physiopathological role. Given the recent discovery that BG shedding can be regulated, TGF-β modulatory properties of BG make it an attractive target for pathologies in which TGF-β action. Despite its reputation as an “accessory” TGF-β receptor, the mice lacking BG (BG-null or TβRIII−/−) exhibited an embryonic lethal phenotype (3), indicating that this coreceptor has essential, not yet identified, cellular functions. Here, we review the current knowledge of BG and discuss future scenarios for BG research and applications.