Bd Hot 19 Rar
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Bd Hot 19 rar
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Abstract: Simple SummaryBreast cancer is among the most common cancers and the leading cause of cancer-related death in women worldwide. Among potential anticancer drugs considered promising in breast cancer treatment are retinoids that act mainly through nuclear retinoic acid receptors (RARs). Clinical trials, however, showed that cancer cells often acquire resistance to retinoid therapy. Therefore, elucidation of underlying mechanisms of retinoid resistance is needed to develop more effective use of retinoids in cancer treatment. In this study, we identify activation of ERK MAP kinase signaling as a novel mechanism for retinoid resistance of breast cancer cells. We show that ERK signaling regulates RAR signaling and inhibition of ERK potentiates tumor-suppressive functions of RARs in breast cancer cells. Moreover, we also reveal that suppression of RAR signaling coincides with activation of ERK signaling in specific subtypes of breast cancers and that these changes are associated with poor prognoses of breast cancer patients. AbstractRetinoic acid (RA) and its synthetic derivatives, retinoids, have been established as promising anticancer agents based on their ability to regulate cell proliferation and survival. Clinical trials, however, have revealed that cancer cells often acquire resistance to retinoid therapy. Therefore, elucidation of underlying mechanisms of retinoid resistance has been considered key to developing more effective use of retinoids in cancer treatment. In this study, we show that constitutive activation of ERK MAP kinase signaling, which is often caused by oncogenic mutations in RAS or RAF genes, suppresses RA receptor (RAR) signaling in breast cancer cells. We show that activation of the ERK pathway suppresses, whereas its inhibition promotes, RA-induced transcriptional activation of RAR and the resultant upregulation of RAR-target genes in breast cancer cells. Importantly, ERK inhibition potentiates the tumor-suppressive activity of RA in breast cancer cells. Moreover, we also reveal that suppression of RAR signaling and activation of ERK signaling are associated with poor prognoses in breast cancer patients and represent hallmarks of specific subtypes of breast cancers, such as basal-like, HER2-enriched and luminal B. These results indicate that ERK-dependent suppression of RAR activity underlies retinoid resistance and is associated with cancer subtypes and patient prognosis in breast cancers.Keywords: breast cancer; retinoids; retinoic acid receptor (RAR); ERK MAP kinase; breast cancer subtypes
Recent studies indicate that angiogenesis is important in the pathogenesis of acute myeloid leukemias (AMLs). Among the various AMLs, the bone marrow angiogenetic response is particularly pronounced in acute promyelocytic leukemia (APL). However, the molecular mechanisms responsible for this angiogenetic response are largely unknown. In the present study, we have explored the role of HHEX, a homeodomain transcription factor, as a possible mediator of the pro-angiogenetic response observed in APL. This transcription factor seems to represent an ideal candidate for this biologic function because it is targeted by PML-RARα, is capable of interaction with PML and PML-RARα, and acts as a regulator of the angiogenetic response.
We used various cellular systems of APL, including primary APL cells and leukemic cells engineered to express PML-RARα, to explore the role of the PML-RARα fusion protein on HHEX expression. Molecular and biochemical techniques have been used to investigate the mechanisms through which PML-RARα downmodulates HHEX and the functional consequences of this downmodulation at the level of the expression of various angiogenetic genes, cell proliferation and differentiation.
Our results show that HHEX expression is clearly downmodulated in APL and that this effect is directly mediated by a repressive targeting of the HHEX gene promoter by PML-RARα. Studies carried out in primary APL cells and in a cell line model of APL with inducible PML-RARα expression directly support the view that this fusion protein through HHEX downmodulation stimulates the expression of various genes involved in angiogenesis and inhibits cell differentiation.
Few studies have explored the expression and a possible deregulation of HHEX in leukemic cells. HHEX was expressed in the large majority of leukemic cell lines and its expression is usually lost when these cell lines are induced to differentiate . In some rare AML patients, it was reported that a specific double translocation involving nucleoporin 98 was fused to the DNA-binding domain of the HHEX transcription factor . The mechanism resulting in leukemia in these patients is not known, but it was proposed that the fusion protein may compete with endogenous HHEX for HHEX targets and may derepress genes normally blocked by HHEX .
Importantly, HHEX was shown to interact with the promyelocytic leukemia protein (PML) in various leukemic cell lines, including the promyelocytic cell line NB4 . Yeast two-hybrid experiments have shown that HHEX was capable of directly interacting with PML across its ring finger domain, which is required for the protein activity in the control of cell growth . Furthermore, HHEX was shown to be able to interact also with the PML-RARα oncoprotein that characterizes acute promyelocytic leukemias (APLs) . According to these observations, it was proposed that disruption not only of PML but also of HHEX functions by PML-RARα fusion protein may play a relevant role in the pathogenesis of APLs . In an attempt to define the mechanism through which PML-RARα blocks myeloid differentiation at the promyelocytic stage, Wang and coworkers have shown that PML-RARα targets promoter regions containing PU.1 consensus and RARE half sites in APL cells . Among the various gene promoters displaying these characteristics, there is also the HHEX promoter, seemingly repressed by PML/RARα binding . PML-RARα-mediated repression of PU.1-mediated transactivation was restored by the addition of all-trans retinoic acid (ATRA). The key functional role of PML-RARα-mediated repression of PU.1 expression and function was carefully confirmed by the same authors in other studies [16, 17].
Given the key role of HHEX in the control of hematopoietic cell differentiation, the targeting of the HHEX gene by the fusion oncoprotein PML-RARα, and the capacity of the HHEX protein to interact with PML and PML-RARα, we sought to investigate the expression and the possible deregulation of HHEX in APLs. In the present study, we have explored the expression and the deregulation of HHEX in APL. Our results indicate that HHEX expression is clearly downmodulated in APLs, while VEGF-A expression is upregulated. The study of an APL cell line model with inducible PML-RARα expression supports the view that this fusion protein significantly downmodulates HHEX expression. The inhibitory effect exerted by PML-RARα on HHEX expression seems to be physio-pathologically relevant to mediate the inhibitory effect on cell differentiation and the pro-angiogenetic effect induced by this fusion protein.
In a previous study, Wang and coworkers have shown that PML/RARα acts as a potent repressor of PU.1-RARE binding sites present at the level of various genes, including HHEX gene; this repression was relieved by all-trans retinoic acid (ATRA) . Given the key role of HHEX as a repressor of various angiogenetic genes and the elevated expression of angiogenetic factors in APL, it seemed of interest to explore in detail the possible consequences of a deregulated HHEX expression in APLs induced by PML-RARα.
Thus, in a first set of experiments, we evaluated HHEX messenger RNA (mRNA) expression by real-time PCR in human CD34+ cells triggered to selective granulocytic differentiation under appropriate and selective cell culture conditions. HHEX was clearly expressed in undifferentiated CD34+ progenitors and its expression progressively and continuously decreased during the process of granulocytic differentiation and maturation (Fig. 1a).
HHEX expression during granulocytic differentiation of normal CB CD34+ cells (a, b) and of primary APL cells (b, c); expression of angiopoietic growth receptors (VEGF-R2 and Tie-2) and ligands (VEGF and angiopoietin-1) in APL primary cells induced to granulocytic differentiation (d, e). Normal CD34+ cells have been purified from cord blood and grown in vitro under cell culture conditions allowing their selective granulocytic differentiation; at different days of culture, cell aliquots were harvested and processed for HHEX expression by real-time PCR analysis. Primary leukemic APL blasts were purified from the bone marrow of an APL patient and grown in vitro in cell culture medium containing 1 μM ATRA, harvested at different times and processed for HHEX, VEGF, VEGF-R2, Tie-2, and angiopoietin-1 expression by real-time PCR
We then compared the level of HHEX mRNA expression observed in either normal CD34+ cells or day 7 granulocytic cells, composed by a majority of normal promyelocytes, with the HHEX mRNA levels observed in fresh diagnostic APL blasts and we observed that HHEX levels were markedly lower in leukemic cells than in their normal counterpart (Fig. 1b). In APL fresh leukemic blasts induced in vitro to terminal granulocytic differentiation, a further decrease of HHEX RNA expression was observed, its expression being virtually absent in terminally differentiated APL cells (i.e., at 120 h after ATRA addition) (Fig. 1c). ATRA also induced a marked downmodulation of the expression of various angiogenetic genes, including VEGF-R2 (Fig. 1d). 041b061a72