RhoA and RhoC differentially modulate estrogen receptor α recruitment, transcriptional activities, and expression in breast cancer cells (MCF-7).

PURPOSE: RhoA and RhoC are closely related, small GTPases that are clearly involved in breast cancer tumorigenesis. Nonetheless, their specific roles in the control of estrogen receptor alpha (ERα) activities have not been elucidated. METHODS: We used siRNA sequences to specifically down-regulate RhoA and RhoC expression in ERα-positive breast adenocarcinoma MCF-7 cells. We then analyzed the consequences of down-regulation on ERα expression, ERα recruitment to the promoters of four target genes, and the mRNA levels of those genes. RESULTS: We demonstrated that RhoA and RhoC clearly and similarly modulated ERα recruitment to the vitellogenin estrogen responsive element (ERE) present in a luciferase reporter gene and to the promoters of progesterone receptor (PR), cathepsin D, and pS2 genes. Besides, RhoA up-regulated the ERE-luciferase reporter gene activity and PR mRNA expression and tended to down-regulate cathepsin D and pS2 mRNA expression. Conversely, RhoC inhibition had no significant effect at the mRNA level. Furthermore, RhoA inhibition, and to a lesser extent RhoC inhibition, increased ERα expression. No alteration in ERα mRNA levels was observed, suggesting potential post-translational control. CONCLUSIONS: Taken together, our results strongly suggest that RhoA and RhoC play different, but clear, roles in ERα signaling. These GTPases are definitely involved, along with RhoB, in ERα recruitment and, to some extent, ERα cofactor balance. We hypothesize a differential role of RhoA in breast cancer tumors that depend on hormone status.

RhoB modifies estrogen responses in breast cancer cells by influencing expression of the estrogen receptor.

INTRODUCTION: RhoB has been reported to exert positive and negative effects on cancer pathophysiology but an understanding of its role in breast cancer remains incomplete. Analysis of data from the Oncomine database showed a positive correlation between RhoB expression and positivity for both estrogen receptor alpha (ERα) and progesterone receptor (PR). METHODS: This finding was validated by our analysis of a tissue microarray constructed from a cohort of 113 patients and then investigated in human cell models. RESULTS: We found that RhoB expression in tissue was strongly correlated with ERα and PR expression and inversely correlated with tumor grade, tumor size and count of mitosis. In human breast cancer cell lines, RhoB attenuation was associated with reduced expression of both ERα and PR, whereas elevation of RhoB was found to be associated with ERα overexpression. Mechanistic investigations suggested that RhoB modulates ERα expression, controlling both its protein and mRNA levels, and that RhoB modulates PR expression by accentuating the recruitment of ERα and other major co-regulators to the promoter of PR gene. A major consequence of RhoB modulation was that RhoB differentially regulated the proliferation of breast cancer cell lines. Interestingly, we documented crosstalk between RhoB and ERα, with estrogen treatment leading to RhoB activation. CONCLUSION: Taken together, our findings offer evidence that in human breast cancer RhoB acts as a positive function to promote expression of ERα and PR in a manner correlated with cell proliferation.

Tipifarnib plus tamoxifen in tamoxifen-resistant metastatic breast cancer: a negative phase II and screening of potential therapeutic markers by proteomic analysis.

PURPOSE: Tipifarnib, a farnesyltransferase inhibitor, has antitumor activity in heavily pretreated metastatic breast cancer patients. Preclinical data suggest that FTIs could restore tamoxifen responsiveness in tamoxifen-resistant disease. Thus, combining FTIs and tamoxifen may be a promising clinical approach after relapse or progression on tamoxifen. EXPERIMENTAL DESIGN: Postmenopausal patients with measurable estrogen receptor- and/or progesterone receptor-expressing metastatic breast cancers were enrolled. Only patients with disease progression on tamoxifen were eligible, but there was no limitation regarding prior chemotherapy or hormone therapy regimens. Patients were immediately treated with 300 mg (n = 12) or 200 mg (n = 10) tipifarnib twice daily for 21 of 28-day cycles plus tamoxifen once daily. Serum was collected at baseline and after 8 weeks of treatment to enable proteomic comparison and identify possible predictive response markers. RESULTS: Twenty patients were enrolled and evaluated for efficacy: one patient had an objective response (liver metastasis) and nine had stable disease after 6 months for a clinical benefit rate of 50%; median duration of benefit was 10.3 (range, 7.4-20.2) months. The proteomic analysis by SELDI-TOF and LTQ-FT-Orbitrap identified a known peptide of fibrinogen alpha, the intensity of which was significantly increased in patients with progression compared with patients who benefited from the combined treatment after 8 weeks. CONCLUSIONS: Because the primary end point of efficacy (three objective responses) was not achieved, the study is negative. Nevertheless, the identified peptide could be of interest in discriminating, at 8 weeks of treatment, responders from nonresponders.

Functional platelet-activating factor receptors in immature forms of leukemic blasts.

Platelet-activating factor (PAF) is a phospholipid mediator with potent immunoregulatory activities on mature leukocytes. PAF modulates leukocyte cytosolic Ca2+ concentration ([Ca2+]i) through a Gq mediated pathway. We highlight, for the first time, Gq transcripts, PAF receptor (PAF-R) transcripts and protein in blast cells of acute myeloid (AML) and lymphoid (ALL) leukemia patients. PAF stimulated [Ca2+]i in leukemic blast cells; PAF effects being prevented by a specific PAF-R antagonist. In conclusion, functional PAF-R are present in blast cells of patients with acute leukemia; a result that could be of physiologic importance regarding the important effect of PAF on leukocytes maturation and functions.

Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: an ex vivo analysis.

BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.

PGE(2) receptor subtype functionality on immature forms of human leukemic blasts.

The ability of prostaglandin E2 (PGE2) to regulate the immune system is well documented. PGE2 effects are mediated through interactions with four distinct membrane EP receptors (EP(1-4)). We investigated, for the first time, the functionality of EP receptors on immature forms of blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. RT-PCR experiments documented the presence of the four EP receptor subtype transcripts in leukemic blasts of AML M0, AML M1, AML M2 and ALL patients. Western blot analysis only documented the presence of the EP2 receptor. Functional assays (cAMP production, calcium flux) confirmed Western blot results, i.e., the presence of functional EP2 receptors. Results of the present study suggest that the mechanism used by PGE2 to influence blast physiology is mediated through the EP2 receptor subtype, and subsequently through a cAMP-elevating effect. Results obtained with M0-2 subtypes have to be necessarily extended to more differentiated phenotype.