Lipin-1 regulates cancer cell phenotype and is a potential target to amplify the effect of metabolic inhibitors

An increased lipogenesis was reported in various cancers and its inhibition represses proliferation and induces apoptosis of cancer cells while barely affecting most normal cells. The family of lipins encompasses three members: lipin-1, -2 and -3. All exhibit dual functions as enzymes, catalyzing the de-phosphorylation of phosphatidic acid to diacylglycerol, and as co-transcriptional regulators. Thus, they are able to regulate lipid homeostasis at several nodal points. However, no studies addressed the involvement of these proteins in cancer progression neither in vitro nor in vivo. During this PhD work, we first investigated the importance of lipin-1 on prostate cancer cell phenotype. We showed that lipin-1 expression is increased in various cancer cell types both in vitro and in vivo in human prostate tumor samples. The specific inhibition of lipin-1 in prostate and breast cancer cells demonstrated its critical importance for cell proliferation and migration through deregulation of several intracellular signaling pathways. This study demonstrated for the first time that the targeting of lipin-1 is a potential new anti-cancer strategy that could be used alone or in combination with drugs like rapamycin. During the course of these investigations, we also observed a compensatory regulation between lipin-1 and lipin-2 preventing the complete inhibition of lipins enzymatic activity when using a siRNA approach. Therefore, we focused our interest on inhibiting all lipins through treatment with a pharmacological inhibitor, propranolol. This drug also induced a blockade of the late phases of autophagy, likely because the inhibition of all lipins decreases diacylglycerol synthesis, which is required for the maturation of autolysosomes. Autophagy is a pro-survival mechanism induced by several stresses or by anticancer agents. In the case of the glycolysis inhibitor 2DG, the induction of autophagy limits its anticancer effects in monotherapy. In the light of their individual specific effects on cancer cells, we hypothesized that the combination of propranolol and 2DG might exert a more than additive toxic effect on cancer cells. In this second study, we report that combined treatment is effective on preventing prostate cancer cell proliferation, inducing cell apoptosis, altering the function of several organelles as mitochondria, endoplasmic reticulum and Golgi apparatus in vitro but also in suppressing tumor growth in vivo. Altogether, our data suggest that lipins are key players involved in cancer progression. Targeting lipins, alone or in combination with other treatments, could open new avenues in anticancer therapy

Lipin-1 regulates cancer cell phenotype and is a potential target to potentiate rapamycin treatment

peer reviewedLipogenesis inhibition was reported to induce apoptosis and repress proliferation of cancer cells while barely affecting normal cells. Lipins exhibit dual function as enzymes catalyzing the dephosphorylation of phosphatidic acid to diacylglycerol and as co-transcriptional regulators. Thus, they are able to regulate lipid homeostasis at several nodal points. Here, we show that lipin-1 is up-regulated in several cancer cell lines and overexpressed in 50 % of high grade prostate cancers. The proliferation of prostate and breast cancer cells, but not of non-tumorigenic cells, was repressed upon lipin-1 knock-down. Lipin-1 depletion also decreased cancer cell migration through RhoA activation. Lipin-1 silencing did not significantly affect global lipid synthesis but enhanced the cellular concentration of phosphatidic acid. In parallel, autophagy was induced while AKT and ribosomal protein S6 phosphorylation were repressed. We also observed a compensatory regulation between lipin-1 and lipin-2 and demonstrated that their co-silencing aggravates the phenotype induced by lipin-1 silencing alone. Most interestingly, lipin-1 depletion or lipins inhibition with propranolol sensitized cancer cells to rapamycin. These data indicate that lipin-1 controls main cellular processes involved in cancer progression and that its targeting, alone or in combination with other treatments, could open new avenues in anticancer therapy.Analyse du rôle de la lipin-1 (phosphatidic acid phosphatase-1) dans la progression tumorale et de sa régulation transcriptionnelle et post-traductionnelle par les RhoGTPase

Propranolol sensitizes prostate cancer cells to glucose metabolism inhibition and prevents cancer progression.

Propranolol, a widely used non-selective beta-adrenergic receptor blocker, was recently shown to display anticancer properties. Its potential to synergize with certain drugs has been also outlined. However, it is necessary to take into account all the properties of propranolol to select a drug that could be efficiently combined with. Propranolol was reported to block the late phase of autophagy. Hence, we hypothesized that in condition enhancing autophagy flux, cancer cells should be especially sensitive to propranolol. 2DG, a glycolysis inhibitor, is an anti-tumor agent having limited effect in monotherapy notably due to induction of pro-survival autophagy. Here, we report that treatment of cancer cells with propranolol in combination with the glycolysis inhibitor 2DG induced a massive accumulation of autophagosome due to autophagy blockade. The propranolol +2DG treatment efficiently prevents prostate cancer cell proliferation, induces cell apoptosis, alters mitochondrial morphology, inhibits mitochondrial bioenergetics and aggravates ER stress in vitro and also suppresses tumor growth in vivo. Our study underlines for the first time the interest to take advantage of the ability of propranolol to inhibit autophagy to design new anti-cancer therapies.Roles of lipins in cancer progression and analysis of their potential as targets for cancer therap

Expansion of CD16+ CD56+ NK cells in vericyte® NK cell growth medium

Natural Killer (NK) cells play a key role in host resistance to virus and tumour. These cells are potent killers of virus infected and tumour cells via a direct recognition of the target by activation receptor such as NKG2D or by inducing Fcγ receptor (FcγRIII, CD16) mediated antibody dependent cellular cytotoxicity (ADCC). Current NK cell-based cancer immunotherapy aims to produce large amounts of functional NK cells, unfortunately most culture media used for NK cell expansion induced the downregulation of CD16 on NK cells. Here, we tested the impact of a new NK cell growth medium (Vericyte® from Medicyte) on CD16 expression. Sorted NK cells and peripheral blood mononuclear cells (PBMC) were cultivated in vericyte® NK cell growth medium and cells issued from these cultures were characterized in term of expansion and phenotype at several time points. After 5 days of culture, an expansion of both NK cells and PBMC was observed and maintained at least until day 20 of culture. In PBMC cultures, we observe only a small preferential NK cell growth since NK cells were around 5-10% at beginning of the culture and this percentage increased to 15% at the end of the culture. However, these cells showed a high proliferative potential when we started the culture with sorted NK cells (the proportion of contaminant cells remain low, under 5%). NK cells expressed CD56 and NKp46 and interestingly after a decreased expression of CD16 on the cell surface at day 3, this receptor was up regulated and most of the cells are CD56bright CD16bright from day 7 to day 12. According FACS FCS/SSC dot plot, NK cells acquired morphology of large activated lymphocytes and some of them expressed activation markers such CD25. Finally, these cells were able to kill efficiently tumour cell line K562. Thus our data show that vericyte® NK cell growth medium allows the expansion of functional CD16+CD56+ NK cells. Cytokine production and ADCC function are under investigation

La régulation induite par l'acidose G0S2 adipocytaire favorise le dialogue entre les adipocytes et les cellules cancéreuses ainsi que la progression tumorale.

Bidirectional interactions between cancer cells and their microenvironment govern tumor growth and development. Among the stromal cells present in this microenvironment, adipocytes were reported to up-regulate cancer cell migration and invasion by providing fatty acids to tumor cells. Also, tumor cells were reported to alter adipocyte phenotype notably by increasing lipolysis. Here, we aimed at identifying key adipocyte genes involved in the crosstalk between tumor cells and adipocytes. Adipose triglyceride lipase (ATGL), CGI-58 (its activator) and G0S2 (one of its inhibitors) are key actors of the first and rate-limiting step of lipolysis. Among them, we observed that G0S2 is the most strongly repressed in adipocytes upon co-culture with cancer cells. During these experiments, we noticed an acidification of the culture medium which we demonstrated to be the main driver of cancer cell-induced lipolysis. This prompted us to evaluate the effect of acidosis on gene expression in adipocytes. G0S2 expression was repressed upon acidification to pH values commonly observed in the tumor microenvironment. We demonstrated that the inhibition of G0S2 expression can be reversed by PPARgamma activators, and showed, by chromatin immunoprecipitation experiments, that CEBP/alpha is involved in G0S2 regulation. To further analyze the role of G0S2, we generated adipocytes expressing G0S2 in a doxycycline-dependent way. We observed that re-expression of G0S2 in adipocyte was sufficient to repress lipolysis, to decrease the pro-migratory effect exerted by adipocyte on cancer cells and, most importantly, to antagonize the pro-survival effect exerted by adipocytes on cancer cells treated with the chemotherapeutic agent doxorubicin. In vivo, the specific overexpression of G0S2 in the adipose tissues of MMTV-PyMT mice decreased tumor growth and metastasis formation. Our results highlight new cellular regulations induced by tumor acidosis and describe how they alter the crosstalk between cancer cells and their microenvironment as well as tumor progression

Le rôle de G0S2 adipocytaire dans le dialogue entre les cellules cancéreuses et les adipocytes

Cancer cells are intermingled within a microenvironment consisting of stromal cells, immune cells and a large assortment of extracellular matrix proteins. Bidirectional interactions between cancer cells and their microenvironment govern tumor growth and development. Among the stromal cells present in this microenvironment, adipocytes were reported to up-regulate cancer cells migration and invasion by providing fatty acids to tumor cells. Also, tumor cells were reported to alter adipocyte phenotype notably by increasing lipolysis. Here, we aimed at identifying key adipocyte genes involved in the crosstalk between tumor cells and adipocytes. Adipose triglyceride lipase (ATGL), CGI-58 (its activator) and G0S2 (one of its inhibitors) are key actors of the first and rate-limiting step of lipolysis. Among them, we observed that G0S2 is the most strongly repressed in adipocytes upon co-culture with cancer cells. During these experiments, we noticed an acidification of the culture medium which we demonstrated to be the main driver of cancer cells-induced lipolysis. We further established that slight acidification of culture medium to pH values similar to those encountered in tumors was sufficient to repress the expression of G0S2 in adipocytes, that this inhibition can be reversed by PPARɣ activators and that CEBPα is involved in this regulation (as revealed through chromatin immunoprecipitation assay). To better characterize the potential implication of G0S2 in cancer progression, we generated adipocytes expressing G0S2 in a doxycycline-dependent way. We observed that re-expression of G0S2 in adipocyte was sufficient to repress lipolysis, to decrease their capacity to stimulate tumor cell migration and to antagonize their pro-survival effect on cancer cells treated with the chemotherapeutic agent doxorubicin. In order to verify in vivo the relevance of our in vitro data, we decided to use MMTV-PyMT mice because they develop spontaneous tumors in the mammary glands, a tissue rich in adipocytes. PyMT mice overexpressing G0S2 specifically in adipose tissue (PyMT-aP2G0S2) were generated and compared to control PyMT mice for mammary tumor formation. While tumors developed similarly in both models, the incidence of lung metastases was significantly reduced in the PyMT-aP2G0S2 mice. Our results support a key role for G0S2 in the crosstalk between adipocytes and cancer cells, since the regulation of its expression in adipocytes is sufficient to modulate the phenotype of tumor cells, their resistance to chemotherapeutic agents and their capacity to form metastases in vivo

Lipin-1, a Versatile Regulator of Lipid Homeostasis, Is a Potential Target for Fighting Cancer

The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated

A Novel Physiological Glycosaminoglycan-Deficient Splice Variant of Neuropilin-1 Is Anti-Tumorigenic In Vitro and In Vivo.

Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine612. Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite "pro-tumoral" effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies

Acidosis-induced regulation of adipocyte G0S2 promotes crosstalk between adipocytes and breast cancer cells as well as tumor progression.

peer reviewedBidirectional interactions between cancer cells and their microenvironment govern tumor progression. Among the stromal cells in this microenvironment, adipocytes have been reported to upregulate cancer cell migration and invasion by producing fatty acids. Conversely, cancer cells alter adipocyte phenotype notably via increased lipolysis. We aimed to identify the mechanisms through which cancer cells trigger adipocyte lipolysis and evaluate the functional consequences on cancer progression. Here, we show that cancer cell-induced acidification of the extracellular medium strongly promotes preadipocyte lipolysis through a mechanism that does not involve lipophagy but requires adipose triglyceride lipase (ATGL) activity. This increased lipolysis is triggered mainly by attenuation of the G0/G1 switch gene 2 (G0S2)-induced inhibition of ATGL. G0S2-mediated regulation in preadipocytes affects their communication with breast cancer cells, modifying the phenotype of the cancer cells and increasing their resistance to chemotherapeutic agents in vitro. Furthermore, we demonstrate that the adipocyte-specific overexpression of G0S2 impairs mammary tumor growth and lung metastasis formation in vivo. Our results highlight the importance of acidosis in cancer cell-adipocyte crosstalk and identify G0S2 as the main regulator of cancer-induced lipolysis, regulating tumor establishment and spreading.Roles and regulatory mechanisms of adipocytic lipolysis during tumour progression

NRP1-Δ7 inhibits tumor growth and vascularization <i>in vivo</i>.

<p>(A) 2 x 10⁶ PC3/TR/ NRP1-Δ7 cells were injected in each flank of nude mice (n = 8 mice). The animals were separated in two groups. The expression of the transgene was induced in one group by adding doxycycline (2 mg/ml) in drinking water. After 7 weeks, mice were sacrificed. The tumors were macroscopically observed (A), measured and weighed (B) (n = 15 tumors in control condition and n = 14 tumors in doxycycline-induced mice) and processed for total RNA extraction and measurement of both variants by RT-PCR (C). The relative proportion of NRP1-Δ7 increases from 20 to 50% in presence of dox. (D) Immunological analysis of tumor vascularization by CD31 immunostaining. (E) The number of blood vessels per unit surface was determined and (F) the surface covered by blood vessels was calculated and expressed in %. Results are expressed as the mean ± SD of n = 13 tumors in control condition and n = 10 tumors in doxycycline-induced mice (*: p<0.05, Mann-Whitney U test)—Scale bar = 100 μm.</p