New inhibitor targeting Acyl-CoA synthetase 4 reduces breast and prostate tumor growth, therapeutic resistance and steroidogenesis.

Acyl-CoA synthetase 4 (ACSL4) is an isoenzyme of the fatty acid ligase-coenzyme-A family taking part in arachidonic acid metabolism and steroidogenesis. ACSL4 is involved in the development of tumor aggressiveness in breast and prostate tumors through the regulation of various signal transduction pathways. Here, a bioinformatics analysis shows that the ACSL4 gene expression and proteomic signatures obtained using a cell model was also observed in tumor samples from breast and cancer patients. A well-validated ACSL4 inhibitor, however, has not been reported hindering the full exploration of this promising target and its therapeutic application on cancer and steroidogenesis inhibition. In this study, ACSL4 inhibitor PRGL493 was identified using a homology model for ACSL4 and docking based virtual screening. PRGL493 was then chemically characterized through nuclear magnetic resonance and mass spectroscopy. The inhibitory activity was demonstrated through the inhibition of arachidonic acid transformation into arachidonoyl-CoA using the recombinant enzyme and cellular models. The compound blocked cell proliferation and tumor growth in both breast and prostate cellular and animal models and sensitized tumor cells to chemotherapeutic and hormonal treatment. Moreover, PGRL493 inhibited de novo steroid synthesis in testis and adrenal cells, in a mouse model and in prostate tumor cells. This work provides proof of concept for the potential application of PGRL493 in clinical practice. Also, these findings may prove key to therapies aiming at the control of tumor growth and drug resistance in tumors which express ACSL4 and depend on steroid synthesis.

BRCA1 and BRCA2 mutations and clinical interpretation in 398 ovarian cancer patients: comparison with breast cancer variants in a similar population.

BACKGROUND: Ovarian cancer is the leading cause of death worldwide among gynecologic malignancies. The recent approval of inhibitors of poly (ADP-ribose) polymerase (iPARP) in the treatment of ovarian cancer in the presence of a BRCA1/2 mutation has sparked the analysis of women with such diagnosis, which can further benefit from the detection of carriers in the family. Germline sequence and large rearrangements for BRCA1/2 were tested in 398 consecutive epithelial ovarian cancer (EOC) patients. The aim of this study was to identify the frequency and spectrum of germline BRCA1/2 pathogenic alterations in a cohort of patients with ovarian serous carcinoma, with a view to adequately selecting patients for prevention through family counseling and correlating this frequency with platinum sensitivity as a guidance to identify patients eligible for iPARP in our population. RESULTS: A total of 96 patients carried a pathogenic germline mutation, accounting for an overall 24.1% mutation incidence. Among mutation carriers, BRCA1 showed 62.5% incidence, BRCA2 rendered 36.5%, and one patient exhibited a mutation in both genes. Three pathogenic mutations were recurrent mutations detected five, three, and four times and represented 12.5% of the mutated samples. Worth highlighting, a 50% mutation incidence was detected when breast and ovarian cancer coexisted in the same patient. Novel mutations amounted to 9.4% of the total mutations, as compared to 4.7% in breast cancer. Forty out of 60 BRCA1 mutations were beyond the ovarian cancer cluster region (OCCR), in stark contrast with 22 out of 36 BRCA2 mutations being inside the OCCR. Taken together, germline BRCA1/2 mutations in EOC patients showed a distinct mutational spectrum compared to our previously published data on breast cancer patients. CONCLUSIONS: In sum, our study provides novel data on ovarian BRCA1/2 mutation prevalence worldwide, enhances adequate patient selection for family counseling and prevention, and sheds light on the benefits of iPARP treatment.

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4.

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.

Study of the Genetic Variants in BRCA1/2 and Non-BRCA Genes in a Population-Based Cohort of 2155 Breast/Ovary Cancer Patients, Including 443 Triple-Negative Breast Cancer Patients, in Argentina.

Gene/s sequencing in hereditary breast/ovary cancer (HBOC) in routine diagnosis is challenged by the analysis of panels. We aim to report a retrospective analysis of BRCA1/2 and non-BRCA gene sequencing in patients with breast/ovary cancer (BOC), including triple-negative breast cancer (TNBC), in our population. In total 2155 BOC patients (1900 analyzed in BRCA1/2 and 255 by multigenic panels) gave 372 (17.2.6%) and 107 (24.1%) likely pathogenic/pathogenic variants (LPVs/PVs), including BRCA and non-BRCA genes, for the total and TNBC patients, respectively. When BOC was present in the same proband, a 51.3% rate was found for LPVs/PVs in BRCA1/2. Most of the LPVs/PVs in the panels were in BRCA1/2; non-BRCA gene LPVs/PVs were in CDH1, CHEK2, CDKN2A, MUTYH, NBN, RAD51D, and TP53. TNBC is associated with BRCA1/2 at a higher rate than the rest of the breast cancer types. The more prevalent PVs in BRCA1/2 genes (mostly in BRCA1) do not rule out the importance to panels of genes, although they are certainly far from shedding light on the gap of the 85% predicted linkage association of BOC with BRCA1/2 and are still not elucidated.

Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

Angiotensin II stimulation promotes mitochondrial fusion as a novel mechanism involved in protein kinase compartmentalization and cholesterol transport in human adrenocortical cells.

In steroid-producing cells, cholesterol transport from the outer to the inner mitochondrial membrane is the first and rate-limiting step for the synthesis of all steroid hormones. Cholesterol can be transported into mitochondria by specific mitochondrial protein carriers like the steroidogenic acute regulatory protein (StAR). StAR is phosphorylated by mitochondrial ERK in a cAMP-dependent transduction pathway to achieve maximal steroid production. Mitochondria are highly dynamic organelles that undergo replication, mitophagy and morphology changes, all processes allowed by mitochondrial fusion and fission, known as mitochondrial dynamics. Mitofusin (Mfn) 1 and 2 are GTPases involved in the regulation of fusion, while dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the role of mitochondrial dynamics in neurological and endocrine disorders, little is known about fusion/fission in steroidogenic tissues. In this context, the present work aimed to study the role of angiotensin II (Ang II) in protein subcellular compartmentalization, mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. We demonstrate here that Ang II stimulation promoted the recruitment and activation of PKCε, ERK and its upstream kinase MEK to the mitochondria, all of them essential for steroid synthesis. Moreover, Ang II prompted a shift from punctate to tubular/elongated (fusion) mitochondrial shape, in line with the observation of hormone-dependent upregulation of Mfn2 levels. Concomitantly, mitochondrial Drp1 was diminished, driving mitochondria toward fusion. Moreover, Mfn2 expression is required for StAR, ERK and MEK mitochondrial localization and ultimately for aldosterone synthesis. Collectively, this study provides fresh insights into the importance of hormonal regulation in mitochondrial dynamics as a novel mechanism involved in aldosterone production.

Regulatory mechanisms leading to differential Acyl-CoA synthetase 4 expression in breast cancer cells.

Acyl-CoA synthetase 4 (ACSL4) overexpression plays a causal role in the aggressiveness of triple negative breast cancer. In turn, a negative correlation has been established between ACSL4 and estrogen receptor alpha (ERα) expression. However, the upstream regulatory mechanisms leading to differential ACSL4 expression between triple negative breast cancer and ERα-positive cells remained unknown. We performed the characterization of the human ACSL4 promoter and the identification of transcription factors involved. Deletional analysis demonstrated the proximal 43 base pairs of the promoter are involved in overexpression. By site directed mutagenesis we describe that retinoid-related orphan receptor alpha (RORα), Sp1 and E2F elements are involved in the promoter activity. We established for the first time that estrogen-related receptor alpha (ERRα) is a transcription factor involved in the higher activation of the human ACSL4 promoter in breast cancer cells. Furthermore, a combination of inhibitors of ACSL4 and ERRα produced a synergistic decrease in MDA-MB-231 cell proliferation. We also demonstrated that ERα restoration in triple negative breast cancer cells downregulates ACSL4 expression. The results presented in this manuscript demonstrated transcriptional mechanism is involved in the different expression of ACSL4 in human breast cancer cell lines of different aggressiveness.

New enzymes involved in the mechanism of action of epidermal growth factor in a clonal strain of Leydig tumor cells.

The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.

BRCA1 and BRCA2 Mutations Other Than the Founder Alleles Among Ashkenazi Jewish in the Population of Argentina.

In Ashkenazi Jewish (AJ) high risk families 3 mutations [2 in BRCA1 (c. 68_69del and c.5266dup) and 1 in BRCA2 (c.5946del)] account for the majority of high risk breast and ovarian cancer cases in that ethnic group. Few studies with limited number of genotyped individuals have expanded the spectrum of mutations in both BRCA genes beyond the 3 mutation panel. In this study, 279 high risk individual AJ were counseled at CEMIC (Centro de Educación Médica e Investigaciones Clínicas), and were genotyped first for the 3 recurrent mutation panel followed by Next Generation Sequencing (NGS) of BRCA1 BRCA2 in 76 individuals who tested negative for the first genotyping step. Of 279 probands (259 women), 55 (50 women) harbored one of the 3 mutations (19.7%); Of 76 fully sequenced cases (73 women), 6 (5 women) (7.9%) carried a pathogenic mutation: in BRCA1, c.2728C>T - p.(Gln910*); c.5407-?_(*1_?)del and c.5445G>A - p.(Trp1815*); in BRCA2, c.5351dup - p.(Asn1784Lysfs*3); c.7308del - p.(Asn2436Lysfs*33) and c.9026_9030del - p.(Tyr3009Serfs*7). Of 61 mutation carriers the distribution was as follows: 11 cancer free at the time of genotyping, 34 female breast cancer cases with age range 28-72 years (41.6 ± 9.3), 3 male breast cancer cases with age range 59-75 years (65 ± 7.3), 6 breast and ovarian cancer cases with age range 35-60 years (breast 40.4 ± 5.2; ovary 47.8 ± 7.2) and 7 ovarian cancer cases with age range 41-77 years (60.6 ± 13.3). This information proved highly useful for counseling, treatment, and prevention for the patient and the family. In conclusion comprehensive BRCA1/2 testing in AJ high risk breast ovarian cancer cases adds valuable clinically relevant information in a subset of cases estimated up to 7% and is therefore recommended.

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells.

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.

Tyrosine phosphatases in steroidogenic cells: regulation and function.

In adrenocortical and Leydig cells PKA activation by trophic hormones increases the activity of protein tyrosine phosphatases and also induces the expression of MAP kinase phosphatase 1 (MKP-1), a dual activity protein phosphatase (serine/threonine and tyrosine). This work summarizes the knowledge on the regulation and the role played by cAMP-activated tyrosine phosphatases as well as MKP-1 in the hormonal activation of the acute and chronic phases of steroidogenesis.

Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats.

The aim of the present study was to determine whether the transient receptor potential vanilloid (TRPV1) receptor protein as well as the calcitonin gene-related peptide (CGRP) content could be enhanced after the i.p. administration of 5 mg/kg lipopolysaccharide (LPS) to Sprague-Dawley rats. In tongue tissue, used as a representative model of TRPV1 receptors expression, there was a significant increase in the abundance of TRPV1 receptor protein 6 h after LPS administration. In mesenteric arteries, the density of the CGRP-positive nerves as well as the release of CGRP induced by 10 microM anandamide was also significantly increased 6 h after LPS administration. The relaxant responses induced by anandamide in mesenteric beds isolated from either untreated or LPS-treated rats were abolished after a 2 h exposure to 10 microM capsaicin. Moreover, anandamide-induced relaxations of untreated mesenteries were potentiated by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 0.1 microM), but not by its inactive analogue 4alpha-phorbol (0.1 microM). The potentiation of anandamide effects caused by the PKC activator was accompanied by a significant increase in the overflow of CGRP induced by anandamide in the untreated rats. It is proposed that the overexpression of the TRPV1 receptors and the increased content of CGRP could contribute to the enhancement of anandamide effects during the endotoxemic shock. An eventual phosphorylation event linked to the overflow of CGRP could also participate in the enhanced relaxation caused by anandamide in endotoxemia.

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed.

Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis.

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones.

We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.

The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity.

Acyl-CoA synthetase-4, a new regulator of mTOR and a potential therapeutic target for enhanced estrogen receptor function in receptor-positive and -negative breast cancer.

Although the role of acyl-CoA synthetase 4 (ACSL4) in mediating an aggressive phenotype is well accepted, there is little evidence as to the early steps through which ACSL4 increases tumor growth and progression. In this study, and by means of the stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system (MCF-7 Tet-Off/ACSL4), we identify the mTOR pathway as one of the main specific signatures of ACSL4 expression and demonstrate the partial involvement of the lipoxygenase pathway in the activation of mTOR. The specificity of ACSL4 action on mTOR signaling is also determined by doxycycline inhibition of ACSL4 expression in MCF-7 Tet-Off/ACSL4 cells, by the expression of ACSL4 in the non-aggressive T47D breast cancer cell line and by knocking down this enzyme expression in the MDA-MB-231 breast cancer cells, which constitutively express ACSL4. ACSL4 regulates components of the two complexes of the mTOR pathway (mTORC1/2), along with upstream regulators and substrates.We show that mTOR inhibitor rapamycin and ACSL4 inhibitor rosiglitazone can act in combination to inhibit cell growth. In addition, we demonstrate a synergistic effect on cell growth inhibition by the combination of rosiglitazone and tamoxifen, an estrogen receptor α (ERα) inhibitor. Remarkably, this synergistic effect is also evident in the triple negative MDA-MB-231 cells in vitro and in vivo.These results suggest that ACSL4 could be a target to restore tumor hormone dependence in tumors with poor prognosis for disease-free and overall survival, in which no effective specifically targeted therapy is readily available.

The novel desmopressin analogue [V4Q5]dDAVP inhibits angiogenesis, tumour growth and metastases in vasopressin type 2 receptor-expressing breast cancer models.

Desmopressin (dDAVP) is a safe haemostatic agent with previously reported antitumour activity. It acts as a selective agonist for the V2 vasopressin membrane receptor (V2r) present on tumour cells and microvasculature. The purpose of this study was to evaluate the novel peptide derivative [V4Q5]dDAVP in V2r-expressing preclinical mouse models of breast cancer. We assessed antitumour effects of [V4Q5]dDAVP using human MCF-7 and MDA-MB-231 breast carcinoma cells, as well as the highly metastatic mouse F3II cell line. Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. In order to study the effect of intravenously administered [V4Q5]dDAVP on tumour growth and angiogenesis, breast cancer xenografts were generated in athymic mice. F3II cells were injected into syngeneic mice to evaluate the effect of [V4Q5]dDAVP on spontaneous and experimental metastatic spread. In vitro cytostatic effects of [V4Q5]dDAVP against breast cancer cells were greater than those of dDAVP, and associated with V2r-activated signal transduction and partial cell cycle arrest. In MDA-MB-231 xenografts, [V4Q5]dDAVP (0.3 µg/kg, thrice a week) reduced tumour growth and angiogenesis. Treatment of F3II mammary tumour-bearing immunocompetent mice resulted in complete inhibition of metastatic progression. [V4Q5]dDAVP also displayed greater antimetastatic efficacy than dDAVP on experimental lung colonisation by F3II cells. The novel analogue was well tolerated in preliminary acute toxicology studies, at doses ≥ 300-fold above that required for anti-angiogenic/antimetastatic effects. Our data establish the preclinical activity of [V4Q5]dDAVP in aggressive breast cancer, providing the rationale for further clinical trials.