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.

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.

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.

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.

The functional interaction between Acyl-CoA synthetase 4, 5-lipooxygenase and cyclooxygenase-2 controls tumor growth: a novel therapeutic target.

The acyl-CoA synthetase 4 (ACSL4), which esterify mainly arachidonic acid (AA) into acyl-CoA, is increased in breast, colon and hepatocellular carcinoma. The transfection of MCF-7 cells with ACSL4 cDNA transforms the cells into a highly aggressive phenotype and controls both lipooxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) metabolism of AA, suggesting a causal role of ACSL4 in tumorigenesis. We hypothesized that ACSL4, LOX-5 and COX-2 may constitute potential therapeutic targets for the control of tumor growth. Therefore, the aim of this study was to use a tetracycline Tet-Off system of MCF-7 xenograft model of breast cancer to confirm the effect of ACSL4 overexpression on tumor growth in vivo. We also aim to determine whether a combinatorial inhibition of the ACSL4-LOX-COX-2 pathway affects tumor growth in vivo using a xenograft model based on MDA-MB-231 cells, a highly aggressive breast cancer cell line naturally overexpressing ACSL4. The first novel finding is that stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system of MCF-7 cells resulted in development of growing tumors when injected into nude mice. Tumor xenograft development measured in animals that received doxycycline resulted in tumor growth inhibition. The tumors presented marked nuclear polymorphism, high mitotic index and low expression of estrogen and progesterone receptor. These results demonstrate the transformational capacity of ACSL4 overexpression. We examined the effect of a combination of inhibitors of ACSL4, LOX-5 and COX-2 on MDA-MB-231 tumor xenografts. This treatment markedly reduced tumor growth in doses of these inhibitors that were otherwise ineffective when used alone, indicating a synergistic effect of the compounds. Our results suggest that these enzymes interact functionally and form an integrated system that operates in a concerted manner to regulate tumor growth and consequently may be potential therapeutic targets for the control of proliferation as well as metastatic potential of cancer cells.

Functional interaction between acyl-CoA synthetase 4, lipooxygenases and cyclooxygenase-2 in the aggressive phenotype of breast cancer cells.

The acyl-CoA synthetase 4 (ACSL4) is increased in breast cancer, colon and hepatocellular carcinoma. ACSL4 mainly esterifies arachidonic acid (AA) into arachidonoyl-CoA, reducing free AA intracellular levels, which is in contradiction with the need for AA metabolites in tumorigenesis. Therefore, the causal role of ACSL4 is still not established. This study was undertaken to determine the role of ACSL4 in AA metabolic pathway in breast cancer cells. The first novel finding is that ACSL4 regulates the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin in MDA-MB-231 cells. We also found that ACSL4 is significantly up-regulated in the highly aggressive MDA-MB-231 breast cancer cells. In terms of its overexpression and inhibition, ACSL4 plays a causal role in the control of the aggressive phenotype. These results were confirmed by the increase in the aggressive behaviour of MCF-7 cells stably transfected with a Tet-off ACSL4 vector. Concomitantly, another significant finding was that intramitochondrial AA levels are significantly higher in the aggressive cells. Thus, the esterification of AA by ACSL4 compartmentalizes the release of AA in mitochondria, a mechanism that serves to drive the specific lipooxygenase metabolization of the fatty acid. To our knowledge, this is the first report that ACSL4 expression controls both lipooxygenase and cyclooxygenase metabolism of AA. Thus, this functional interaction represents an integrated system that regulates the proliferating and metastatic potential of cancer cells. Therefore, the development of combinatory therapies that profit from the ACSL4, lipooxygenase and COX-2 synergistic action may allow for lower medication doses and avoidance of side effects.