Carmela Guido

Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production.

Senescent fibroblasts are known to promote tumor growth. However, the exact mechanism remains largely unknown. An important clue comes from recent studies linking autophagy with the onset of senescence. Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST). To test this hypothesis, human fibroblasts immortalized with telomerase (hTERT-BJ1) were stably transfected with autophagy genes (BNIP3, CTSB or ATG16L1). Their overexpression was sufficient to induce a constitutive autophagic phenotype, with features of mitophagy, mitochondrial dysfunction and a shift toward aerobic glycolysis, resulting in L-lactate and ketone body production. Autophagic fibroblasts also showed features of senescence, with increased p21(WAF1/CIP1), a CDK inhibitor, cellular hypertrophy and increased β-galactosidase activity. Thus, we genetically validated the existence of the autophagy-senescence transition. Importantly, autophagic-senescent fibroblasts promoted tumor growth and metastasis, when co-injected with human breast cancer cells, independently of angiogenesis. Autophagic-senescent fibroblasts stimulated mitochondrial metabolism in adjacent cancer cells, when the two cell types were co-cultured, as visualized by MitoTracker staining. In particular, autophagic ATG16L1 fibroblasts, which produced large amounts of ketone bodies (3-hydroxy-butyrate), had the strongest effects and promoted metastasis by up to 11-fold. Conversely, expression of ATG16L1 in epithelial cancer cells inhibited tumor growth, indicating that the effects of autophagy are compartment-specific. Thus, autophagic-senescent fibroblasts metabolically promote tumor growth and metastasis, by paracrine production of high-energy mitochondrial fuels. Our current studies provide genetic support for the importance of “two-compartment tumor metabolism” in driving tumor growth and metastasis via a simple energy transfer mechanism. Finally, β-galactosidase, a known lysosomal enzyme and biomarker of senescence, was localized to the tumor stroma in human breast cancer tissues, providing in vivo support for our hypothesis. Bioinformatic analysis of genome-wide transcriptional profiles from tumor stroma, isolated from human breast cancers, also validated the onset of an autophagy-senescence transition. Taken together, these studies establish a new functional link between host aging, autophagy, the tumor microenvironment and cancer metabolism.

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.

CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth

Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-β signaling, with increased transcription of TGF-β target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-β / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection.

Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as “fuel” for oxidative mitochondrial metabolism. Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach. TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis. Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis, and treatment failure, resulting in poor clinical outcome in breast cancer patients. Thus, this new experimental model system, employing glycolytic fibroblasts, may be highly clinically relevant. These studies also have implications for understanding the role of hydrogen peroxide production in oxidative damage and “host cell aging,” in providing a permissive metabolic microenvironment for promoting and sustaining tumor growth.

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

BackgroundA wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.Methods and ResultsBy RT-PCR, Western blot and Immunofluorescence techniques, we demonstrated that human sperm expresses the 1,25(OH)2D3 receptor (VDR). Besides, 25(OH)D3-1 alpha-hydroxylase, evidenced by Western blot analysis, indicated that in sperm 1,25(OH)2D3 is locally produced, highlighting the potential for autocrine-paracrine responses. 1,25(OH)2D3 through VDR, increased intracellular Ca2+ levels, motility and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability. In sperm, 1,25(OH)2D3 through VDR, reduces triglycerides content concomitantly to the increase of lipase activity. Rapid responses stimulated by 1,25(OH)2D3 have been observed on Akt, MAPK and GSK3 implying that this secosteroid is involved in different sperm signalling pathways.ConclusionOur data extended the role of 1,25(OH)2D3 beyond its conventional physiological actions, paving the way for novel therapeutic opportunities in the treatment of the male reproduction disorders.

Human sperm anatomy: ultrastructural localization of 1α,25-dihydroxyvitamin D3 receptor and its possible role in the human male gamete

Previous studies have suggested that 1α,25‐dihydroxyvitamin D3[1,25(OH)2D3] has a role in reproductive function. Gonadal insufficiencies were observed as a result of 1,25(OH)2D3 deficiency and in 1,25(OH)2D3 receptor (VDR) null mutant mice. To study human sperm anatomy at the molecular level, we first evaluated the ultrastructural localization of VDR by immunogold electron microscopy using a monoclonal antibody against amino acids 344–424 of human VDR, in normozoospermic samples. Intriguingly, VDR was associated predominantly with the cell nucleus. In fact, it is known that VDR is a transcription factor, and that in vitamin‐D‐depleted animals, VDR is largely localized in the cell nucleus. To assess the significance of VDR in the male gamete, we investigated the role of 1,25(OH)2D3/VDR in sperm survival and capacitation. Our results revealed that the action of 1,25(OH)2D3 depended on its concentration because although lower doses induced cholesterol efflux, protein phosphorylation and sperm survival, a higher concentration seemed to be ineffective or did not show an increased effect. These results increase our knowledge of human sperm anatomy at the molecular level and suggest that 1,25(OH)2D3/VDR may have an important role in sperm survival and the acquisition of fertilizing ability.

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene.

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.

Leptin and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival

Several studies have recently investigated the role of leptin, the adipocyte-secreted hormone, in the growth and reproduction of rodents, humans, and domestic animals. The present study was designed to explore the expression of leptin and its receptor in pig spermatozoa. Successful Western blot evidenced a 16 kDa band for leptin and six isoforms, ranging from 120 to 40 kDa, for the leptin receptor. Both leptin and leptin receptor were interestingly located at sperm acrosomal level, suggesting their involvement in the oocyte fertilization events. In fact, both capacitation indexes and acrosin activity were enhanced by leptin, and these effects were reduced by the anti-leptin receptor antibody. Afterwards, we investigated the main transduction pathways regulated by the hormone. Our results showed that, in pig sperm, leptin can trigger the signal transducer and activator of transcription 3, a classical component of cytokine signal transduction pathways, whose expression has not been previously reported in male gamete; in addition it was found constitutively activated. Besides, leptin was able to induce the activation of phosphatidylinositol phosphate kinase 3 and MAP kinase pathways as well as of BCL2, a known antiapoptotic protein. These data address to a role of leptin and its receptor on pig sperm survival. The presence of leptin and its receptor in pig sperm suggests that they, through an autocrine short loop, may induce signal transduction and molecular changes associated with sperm capacitation and survival.

A New Role of Anandamide in Human Sperm: Focus on Metabolism

The endocannabinoid system and the presence of CB1 receptor (CB1‐R) target of the anandamide were identified in human sperm, however the anandamide action in this context needs to be further elucidated. At this purpose we analyzed the effects of anandamide on human sperm capacitation and motility. Afterwards, we focused on lipid and glucose sperm metabolism and also investigated the interrelationship between anandamide and insulin secretion by sperm. By intracellular free Ca2+ content assay and proteins tyrosine phosphorylation, we evidenced that anandamide did not induce capacitation process and a negative effect was obtained on sperm motility. The blockage of CB1‐R by the specific antagonist SR141716 increased both capacitation and sperm motility suggesting an involvement of the CB1‐R in the acquisition of sperm fertilizing activity. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that anandamide exerts a lipogenetic effect on human sperm lipid metabolism. Concerning the glucose metabolism, anandamide increases GSK3 phosphorylation indicating that it is involved in the accumulation of energy substrates. G6PDH activity was not affected by anandamide. Interestingly, AEA is involved in insulin secretion by sperm. As insulin had been demonstrated to be an autocrine factor that triggers capacitation, the endocannabinoid might be inserted in the signaling cascade that induces this process. Altogether these findings highlight a pivotal involvement of the CB1‐R in the control of sperm energy homeostasis and propose a new site of action for endocannabinoids in the control of energy metabolism. J. Cell. Physiol. 221: 147–153, 2009.

Human sperm physiology: Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) influence sperm metabolism and may be involved in the pathophysiology of varicocele-associated male infertility†

The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in “varicocele” sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In “varicocele” sperm, the E2 did not induce energy expenditure. OAT sperm had E2‐responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition. J. Cell. Physiol. 226: 3403–3412, 2011.