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The antitumor effects of pharmacologic inhibitors of angiogenesis are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here, we reevaluated the role of the endogenous antiangiogenic thrombospondin 1 (TSP1) in prostate carcinomas in which angiogenesis is an active process. In xenografted tumors, we observed that TSP1 altogether inhibited angiogenesis and fostered tumor development. Our results show that TSP1 is a potent stimulator of prostate tumor cell migration. This effect required CD36, which also mediates TSP1 antiangiogenic activity, and was mimicked by an antiangiogenic TSP1-derived peptide. As suspected for pharmacologic inhibitors of angiogenesis, the TSP1 capacities to increase hypoxia and to trigger cell migration are thus inherently linked. Importantly, although antiangiogenic TSP1 increases hypoxia in vivo, our data show that, in turn, hypoxia induced TSP1, thus generating a vicious circle in prostate tumors. In radical prostatectomy specimens, we found TSP1 expression significantly associated with invasive tumors and with tumors which eventually recurred. TSP1 may thus help select patients at risk of prostate-specific antigen relapse. Together, the data suggest that intratumor disruption of the hypoxic cycle through TSP1 silencing will limit tumor invasion. Cancer Res; 71(24); 7649–58. ©2011 AACR.

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Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a ‘MitoScore’ signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies. Sarry and colleagues demonstrate that adaptive resistance to venetoclax + cytarabine therapy in acute myeloid leukemia relies on mitochondrial respiration and show that combination with electron transport chain complex inhibitors delays relapse in patient-derived xenograft models in vivo.

Simple Summary Triple-negative breast cancer (TNBC) is associated with a poor prognosis, and the development of better therapeutic strategies is required. Herein, we investigated the role of the anti-angiogenic thrombospondin-1 (TSP1) in TNBC. TSP1 expression in tumor biopsies from TNBC patients was associated with a bad prognosis and a weak content of tumor-infiltrating lymphocytes (TILs). In the 4T1 mouse TNBC model, TSP1 knockdown reduced TGF-β activation and enhanced the content of TILs. Moreover, TSP1 knockdown decreased lung metastasis in syngeneic Balb/c immunocompetent mice but not in immunodeficient nude mice. Finally, TSP1 knockdown enhanced anti-PD-1 immunotherapy efficacy. Thus, targeting TSP1 may be considered as a putative therapeutic strategy in TNBC in combination with immunotherapy. Abstract Triple-negative breast cancer (TNBC) is notoriously aggressive with a high metastatic potential, and targeted therapies are lacking. Using transcriptomic and histologic analysis of TNBC samples, we found that a high expression of thrombospondin-1 (TSP1), a potent endogenous inhibitor of angiogenesis and an activator of latent transforming growth factor beta (TGF-β), is associated with (i) gene signatures of epithelial–mesenchymal transition and TGF-β signaling, (ii) metastasis and (iii) a reduced survival in TNBC patients. In contrast, in tumors expressing low levels of TSP1, gene signatures of interferon gamma (IFN-γ) signaling and lymphocyte activation were enriched. In TNBC biopsies, TSP1 expression inversely correlated with the CD8+ tumor-infiltrating lymphocytes (TILs) content. In the 4T1 metastatic mouse model of TNBC, TSP1 silencing did not affect primary tumor development but, strikingly, impaired metastasis in immunocompetent but not in immunodeficient nude mice. Moreover, TSP1 knockdown increased tumor vascularization and T lymphocyte infiltration and decreased TGF-β activation in immunocompetent mice. Noteworthy was the finding that TSP1 knockdown increased CD8+ TILs and their programmed cell death 1 (PD-1) expression and sensitized 4T1 tumors to anti-PD-1 therapy. TSP1 inhibition might thus represent an innovative targeted approach to impair TGF-β activation and breast cancer cell metastasis and improve lymphocyte infiltration in tumors, and immunotherapy efficacy in TNBC.

To evaluate the expression of CXCR4, its ligand SDF-1, β-catenin and E-cadherin throughout the local tumor microenvironment of prostate cancer.A total of 64 prostate cancer specimens, 24 frozen and 40 paraffin-embedded sections, were obtained from patients treated with radical prostatectomy for clinically localized cancer. Real-time RT-PCR was used for mRNA quantification of CXCR4 and SDF-1 in the tumor center (T), tumor front (F) and distant peritumoral tissue (D). Immunohistochemical analysis was used to investigate the expression patterns of CXCR4, E-cadherin and β-catenin. Clinical records of these patients were studied for follow-up data, and the prognostic value of these molecules' expression was statistically assessed.CXCR4 mRNA and protein were significantly increased at the tumor front as compared to distant tissue or tumor center. In comparison, SDF-1 mRNA level gradually increased from the tumor center to the distant peritumoral tissue. High CXCR4 at the tumor front was associated with high Gleason score. Low SDF-1 at the tumor front was associated with locally advanced cancer and disease recurrence. Moreover, high CXCR4 staining at the tumor front and increased cytosolic E-cadherin expression in the same location was associated with locally advanced disease.CXCR4 seems overexpressed at the tumor front of prostate tumors, where it potentially promotes cell migration toward the SDF-1 centrifugal attracting gradient, as well as epithelial-mesenchymal transition. High CXCR4 and low SDF-1 levels at tumor front were both associated with adverse histological features.

Markers of prostate tumor recurrence after radical prostatectomy are lacking and highly demanded. The androgen receptor (AR) is a nuclear receptor that plays a pivotal role in normal and cancerous prostate tissue. AR interacts with a number of proteins modulating its stability, localization, and activity. To test the hypothesis that an increased expression of AR partners might foster tumor development, we immunopurified AR partners in human tumors xenografted into mice. One of the identified AR partners was the multifunctional enzyme carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the 3 initial steps of pyrimidine biosynthesis. We combined experiments in C4-2, LNCaP, 22RV1, and PC3 human prostate cell lines and analysis of frozen radical prostatectomy samples to study the CAD-AR interaction. We show here that in prostate tumor cells, CAD fosters AR translocation into the nucleus and stimulates its transcriptional activity. Notably, in radical prostatectomy specimens, CAD expression was not correlated with proliferation markers, but a higher CAD mRNA level was associated with local tumor extension (P=0.049) and cancer relapse (P=0.017). These results demonstrate an unsuspected function for a key metabolic enzyme and identify CAD as a potential predictive marker of cancer relapse.

The antiangiogenic extracellular matrix protein thrombospondin-1 (TSP-1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP-1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP-1 in breast cancer, we first used a breast tumorigenesis model in which tumor-associated stromal fibroblasts were engineered to produce high levels of TSP-1. We demonstrate here that stromal TSP-1 delayed human MDA-MB-231/B02 breast tumor growth. However, this delay in MDA-MB-231/B02 tumor growth upon exposure to TSP-1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP-1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP-1. TSP-1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse-free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP-1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP-1, leading to disease progression.

Thrombospondin 1 (TSP1) is a multifunctional protein able to activate TGFβ and to inhibit angiogenesis in vivo. Although usually thought of as an inhibitor of tumor growth, TSP1 may sometimes be present at high levels during tumor progression, suggesting that tumors can eventually overcome their anti-tumor effects. Using a tet-repressible expression system, we demonstrate that murine TSP1 delayed the onset of tumor growth when produced in the tumor bed by rat fibrosarcoma tumor cells or by stromal fibroblasts coinjected with unmodified C6 glioma tumor cells. Yet upon prolonged exposure to TSP1, tumors came to grow at the same rate in the presence as in the absence of TSP1 and transplantation experiments showed that they had become insensitive to inhibition by TSP1 in both syngeneic and immune compromised hosts. Tumor resistance to TSP1 developed as a result of the in vivo outgrowth of pre-existing tumor cell variants that (1) secreted increased amounts of angiogenic factors that counterbalanced the inhibitory effect of TSP1 on neovascularization and (2) grew more efficiently in the presence of TSP1-activated TGFβ. These results indicate that prolonged and continuous local delivery of a single multifunctional angiogenesis inhibitor like TSP1 to fast-growing tumors can lead to tumor resistance in vivo by fostering the outgrowth of subpopulations that are a by-product of the genetic instability of the tumor cells themselves.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. We identified c-Jun-upregulated cDNA clones homologous to the tenascin-C gene by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induced tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis was repressed after stable transformation by c-Jun compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. Furthermore, as demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. We previously described two other extracellular matrix proteins, SPARC and thrombospondin-1, as c-Jun targets. Thus, our results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation.

The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Jun-encoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth.

The antitumor effects of pharmacologic inhibitors of angiogenesis are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here, we reevaluated the role of the endogenous antiangiogenic thrombospondin 1 (TSP1) in prostate carcinomas in which angiogenesis is an active process. In xenografted tumors, we observed that TSP1 altogether inhibited angiogenesis and fostered tumor development. Our results show that TSP1 is a potent stimulator of prostate tumor cell migration. This effect required CD36, which also mediates TSP1 antiangiogenic activity, and was mimicked by an antiangiogenic TSP1-derived peptide. As suspected for pharmacologic inhibitors of angiogenesis, the TSP1 capacities to increase hypoxia and to trigger cell migration are thus inherently linked. Importantly, although antiangiogenic TSP1 increases hypoxia in vivo, our data show that, in turn, hypoxia induced TSP1, thus generating a vicious circle in prostate tumors. In radical prostatectomy specimens, we found TSP1 expression significantly associated with invasive tumors and with tumors which eventually recurred. TSP1 may thus help select patients at risk of prostate-specific antigen relapse. Together, the data suggest that intratumor disruption of the hypoxic cycle through TSP1 silencing will limit tumor invasion. Cancer Res; 71(24); 7649–58. ©2011 AACR.

Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA–mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa. Cancer Res; 70(3); 1225–35

Background Netrin-1 may promote colorectal and breast tumorigenesis, by inhibiting apoptosis induced by its dependence receptors, deleted in colorectal cancer (DCC) and uncoordinated-5-homolog (UNC5H). The status of netrin-1 and its receptors in non – small cell lung cancer (NSCLC) was unknown. Methods The levels of netrin-1 and its receptors were analyzed in a panel of 92 NSCLC and 25 human lung cancer cell lines by quantitative reverse transcription – polymerase chain reaction and immunohistochemistry. In lung cancer cell lines that express netrin-1, the expression of netrin-1 was inhibited by using small interfering RNA (siRNA), or interference with netrin-1 was performed by treatment with a decoy recombinant DCC ectodomain protein (DCC-5Fbn). Cell death was monitored with a trypan blue exclusion assay or by measuring caspase-3 activity. The effect of netrin-1 interference on tumor growth was analyzed by DCC5Fbn intratumoral or netrin-1 siRNA intraperitoneal injection in mice engrafted with lung cancer cell lines. All statistical tests were two-sided. Results High levels of netrin-1 were found in 43 of the 92 NSCLC tumor samples (47%). Interference with netrin-1 in human lung cancer cell lines was associated with UNC5H-mediated cell death in vitro (percentage of cell death in untreated and in DCC-5Fbn – treated cells = 8% and 26%, respectively, difference = 18%, 95% confidence interval [CI] = 10% to 26%; P = .049) and with lung tumor growth inhibition and/or regression in xenografted nude mice (12 mice in DCC-5Fbn – treated group and 13 mice in control group). Mean volume of control and DCC-5Fbn – treated tumors on day 46 was 489 and 84 mm 3 , respectively (difference = 404 mm 3 , 95% CI = 145 to 664 mm 3 ; P < .001). Conclusions Almost half of the NSCLC tissue samples examined expressed high levels of netrin-1. Extracellular targeting of the interaction between netrin-1 and UNC5H may be a promising therapeutic approach for NSCLCs that express netrin-1.

Background Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. Methodology/Principal Findings To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. Conclusions/Significance Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5'UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process.

Contains fulltext : 47464thebault.pdf (Publisher’s version ) (Closed access) TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

In the past few years, several laboratories have developed antiangiogenic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate.

Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.

International audience; It may be postulated that the encephalopathy induced by the human immunodeficiency virus HIV-1, in particular, the characteristic "myelin pallor," may result from binding of the envelope glycoprotein gp120 to galactosylceramide and/or its metabolite sulfatide in the plasma membrane of oligodendrocytes, the myelin forming cells in the central nervous system. (1) gp120 has been reported to have a high affinity for these molecules in vitro. (2) The binding of antibodies to these molecules increases intracellular free calcium levels, which may be cytotoxic. (3) The binding of gp120 to the CD4 receptor in the immune system has the same effect. We have investigated the binding of gp120 to rat oligodendrocytes in vitro by indirect immunofluorescence and have monitored changes in intracellular free calcium with the calcium-sensitive dye INDO-1, in individual oligodendrocytes exposed to the glycoprotein. Antibodies against galatosylceramide and sulfatide bound to the cell membrane, but gp120 did not. The antibodies also increased intracellular free calcium levels in the oligodendrocytes, whereas gp120 did not. It, therefore, seems highly improbable that the demyelination observed during HIV encephalopathy is a direct cytotoxic effect of gp120 on oligodendrocytes.

Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape--frequency and delay--are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists (bicalutamide or flutamide). Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RR(intermittent): 14.5, RR(complete blockade): 6.5 and 1.35). All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.

Metastasis are responsible for 90% of cases of patients death and so represent a major therapeutic challenge in oncology. Anti-angiogenic treatments, which inhibit tumor vascularization and increase hypoxia, have limited clinical efficiency. This inefficiency could be a consequence of hypoxia. Indeed, links have been established between hypoxia and: metabolic changes in tumor cells, increase in cancer stem cells, epithelial-mesenchymal transition and resistance to treatment. Experimental and clinical evidence also suggest that hypoxia may directly induce metastatic dissemination. These observations led us to reassess the role of hypoxia in metastatic dissemination by focusing on the study of the first identified endogenous angiogenic inhibitor, the Thrombospondin 1 (TSP1). TSP1 is also an activator of latent TGFbéta. The aim of this work was to study the role of TSP1 in tumor progression and dissemination of metastatic breast cancer. In clinic, we observed that the expression of TSP1 is correlated with markers of aggressiveness and that it increases with tumor grade. In an orthotopic model of metastatic breast tumors, we have shown that inhibition of TSP1 strongly reduce EMT, invasion and lung metastasis meanwhile the vascularization of the primary tumor is densified and more functional, thus reducing hypoxia. To determine the role of TSP1 in the occurrence of metastasis, we generate point mutations of this gene in TGFbéta activation domain or in the binding domain to CD36 that predominantly mediates the anti-angiogenic properties of TSP1. Mutations of either of these sequences result in a drastic decrease in lung metastasis, which confirms the importance of TGFbéta in metastatic dissemination. Our results also demonstrate a direct link between hypoxia and metastasis: the inactivation of anti-angiogenic properties of TSP1, which increases tumor perfusion and decreases hypoxia without affecting primary tumor growth, blocks metastatic dissemination even though the cells have the ability to migrate. This work led us to propose a new therapeutic concept aiming to normalize tumor vascularization and reduce hypoxia by an "anti-antiangiogenic" strategy based on TSP1 inhibition. This strategy should inhibit metastatic spreading, promote the diffusion of chemotherapeutic treatments and increase the efficiency of radiotherapy, dependent on oxygen presence.; Les métastases sont à l'origine dans 90% des cas du décès des patients et sont donc un enjeu thérapeutique majeur. Les traitements anti-angiogéniques qui inhibent la vascularisation tumorale et augmentent l'hypoxie ont une efficacité limitée en clinique. Des liens ont été établis entre l'hypoxie et le changement métabolique des cellules tumorales, l'augmentation des cellules souches cancéreuses, la transition épithélio-mésenchymateuse et la résistance aux traitements mais également la dissémination métastatique. Ces observations nous ont poussés à réévaluer le rôle de l'hypoxie dans la dissémination métastatique du cancer du sein, en nous focalisant sur l'étude du premier inhibiteur endogène de l'angiogenèse identifié, la Thrombospondine 1 (TSP1). En clinique, nous avons observé que l'expression de la TSP1 est corrélée aux marqueurs d'agressivité et qu'elle augmente avec le grade tumoral. Dans un modèle orthotopique de tumeurs mammaires métastatiques, nous avons montré que l'inhibition de la TSP1 réduit fortement l'EMT, l'invasion et les métastases pulmonaires alors même que la vascularisation de la tumeur primaire est densifiée et plus fonctionnelle, réduisant l'hypoxie. Pour déterminer le rôle de la TSP1 dans la survenue des métastases, nous avons effectué des mutations ponctuelles de ce gène dans le domaine d'activation du TGFbéta ou dans celui de la liaison au CD36 qui médie majoritairement les propriétés anti-angiogéniques de la TSP1. La mutation de l'une ou l'autre de ces séquences entraîne une diminution drastique des métastases pulmonaires. Ce travail nous conduit à proposer un nouveau concept thérapeutique, visant à normaliser la vascularisation tumorale et à réduire l'hypoxie par une stratégie "anti-antiangiogénique" basée sur l'inhibition de la TSP1. Cette stratégie devrait inhiber la dissémination métastatique et favoriser la pénétration des traitements chimiothérapeutiques et augmenter l'efficacité de la radiothérapie, dépendante de la présence d'oxygène.

In addition to the pivotal role of the androgen receptor, the immediate tumor microenvironment plays an essential role in prostate cancer progression. Local hypoxia, through multiple regulation mechanisms, may be implicated in the migration of tumor cells and dissemination. The aim of my thesis was to identify new therapeutic targets and prognostic markers for these cancers. I first participated in the identification of a new partner of the androgen receptor, the protein CAD that is a key enzyme of pyrimidine synthesis. We also studied the role of the endogenous anti-angiogenic thrombospondin-1 (TSP-1) in prostate cancer progression. Although anti-angiogenic activity is usually matched with tumor inhibition, we showed that TSP-1 exerted protumoral effects by stimulating cell migration. These observations led me to study the hypoxia-induced CXCR4/SDF-1 axis that is known to promote cell migration through the extra-cellular matrix. We showed on human tissue that CXCR4 was overexpressed in the tumor front of localized and locally advanced prostate cancers, and that its expression was associated with an epithelial mesenchymal transition. An increasing gradient of SDF-1 was observed from the tumor center to the distant peritumoral tissue, potentially attracting CXCR4-expressing tumor cells of the tumor front. CXCR4 overexpression in the tumor front, as well as SDF1 gradient, were associated with prognosis., Parallèlement au rôle central du récepteur aux androgènes, l’environnement tumoral immédiat exerce aussi une action majeure sur la progression du cancer de la prostate. L’hypoxie locale, par le biais de régulations multiples, serait impliquée dans la migration cellulaire et la dissémination tumorale. L’objectif de ma thèse a été d’identifier de nouvelles cibles thérapeutiques et de nouveaux marqueurs pronostiques pour ces cancers. J’ai tout d’abord participé à l’identification d’un partenaire du récepteur aux androgènes, la protéine CAD, enzyme clé de la synthèse des pyrimidines. Parallèlement, nous nous sommes intéressés au rôle d’un anti-angiogénique endogène dans la progression du cancer de la prostate, la thrombospondine-1 (TSP-1). Bien que l’activité anti angiogénique soit souvent considérée comme anti-tumorale, nous avons mis en évidence le caractère protumoral de la TSP-1, par son action promigratoire sur les cellules tumorales. Ces travaux m’ont conduits à étudier l’axe CXCR4/SDF-1, régulé en partie par l’hypoxie et stimulant la migration cellulaire. Nous avons montré sur tissu humain que CXCR4 était exprimé principalement au niveau du front tumoral des cancers localisés et localement avancés, et que son expression était associée à une transition épithélio-mésenchymateuse. SDF-1 était surexprimée selon un gradient croissant allant du centre des tumeurs vers le tissu péritumoral distant, exerçant possiblement un chimiotactisme sur les cellules du front tumoral. La surexpression de CXCR4 au front tumoral ainsi que le gradient de SDF1 étaient associés au pronostic.

Prognosis is poor once tumors developed metastasis, and overall survival has been only modestly improved by the development of drugs inhibiting angiogenesis. The poor response of certain types of cancer to therapies that target tumor angiogenesis, including prostate and breast carcinomas, is due to the development of an evasion and to the acceleration of the metastatic process noted in some preclinical and clinical studies. The objective of this thesis was to study the role of an endogenous anti-angiogenic factor, thrombospondin 1 (TSP1), in the process of invasion and metastasis of prostate and breast carcinomas. Our laboratory has previously shown that microvessel density is inversely correlated with the expression of TSP1 in primary stages of breast and prostate carcinomas. However, this correlation is lost in advanced cancers, where TSP1 expression becomes associated with poor prognosis. We show that TSP1 stimulates a set of processes involved in tumor progression, including cell migration and invasion, extravasation and metastatic dissemination. We also demonstrate that hypoxia induced by an inhibition of angiogenesis resulting from the activity of endogenous or pharmacological molecules is a major activator of invasion and metastasis. In addition, we demonstrate that TSP1 is a poor prognostic factor since its expression is correlated with increased invasion and relapse in patients with androgen-resistant prostate cancer. In conclusion, our results suggest that the expression of TSP1 could be a predictor of invasion and metastasis occurrence, and that targeting TSP1 could be a great therapeutic potential to block these processes.; Les métastases représentent l’étape ultime de la progression tumorale. Les traitements inhibiteurs de l’angiogenèse offrent de nouvelles perspectives thérapeutiques, notamment pour les stades invasifs, mais cependant ils n’apportent qu’une prolongation modeste de la survie des patients. La faible réponse de certains types de cancers aux traitements ciblant l’angiogenèse tumorale, comme le carcinome de la prostate chez l’homme ou du sein chez la femme, est due au développement de résistances tumorales d’une part, et à l’accélération du processus métastatique mise en évidence par certaines études précliniques et cliniques d’autre part. L’objectif de ce travail de thèse a été d’étudier le rôle d’une protéine anti-angiogénique endogène, la thrombospondine 1 (TSP1), dans l’invasion et la dissémination métastatique dans le carcinome de la prostate et du sein. Notre laboratoire a montré que la densité microvasculaire est inversement corrélée à l’expression de la TSP1 dans les stades précoces de ces carcinomes. Cependant, cette corrélation est perdue aux stades avancés où l’expression de la TSP1 devient associée à un mauvais pronostic. Les travaux de ma thèse ont permis de montrer que la TSP1 stimule un ensemble de processus intervenant dans la progression tumorale, dont la migration et l’invasion cellulaires, l’extravasation et la dissémination métastatique. Nous avons aussi pu mettre en évidence que l’hypoxie induite par l’inhibition de l’angiogenèse est un activateur majeur de l’invasion et de la formation de métastases, que cette hypoxie soit produite par des molécules endogènes comme la TSP1 ou par des inhibiteurs pharmacologiques de ce processus. De plus, nous démontrons que la TSP1 est un facteur de mauvais pronostic puisque son expression est corrélée avec l’augmentation de l’invasion et de la rechute des patients atteints de cancers de la prostate androgéno-résistants. En conclusion, nos résultats suggèrent que l’expression de la TSP1 pourrait être un facteur prédictif de l’invasion et de l’occurrence de métastases, et que le ciblage de la TSP1 présente un fort potentiel thérapeutique pour bloquer à terme ces processus.

Prognosis is poor once tumors developed metastasis, and overall survival has been only modestly improved by the development of drugs inhibiting angiogenesis. The poor response of certain types of cancer to therapies that target tumor angiogenesis, including prostate and breast carcinomas, is due to the development of an evasion and to the acceleration of the metastatic process noted in some preclinical and clinical studies. The objective of this thesis was to study the role of an endogenous anti-angiogenic factor, thrombospondin 1 (TSP1), in the process of invasion and metastasis of prostate and breast carcinomas. Our laboratory has previously shown that microvessel density is inversely correlated with the expression of TSP1 in primary stages of breast and prostate carcinomas. However, this correlation is lost in advanced cancers, where TSP1 expression becomes associated with poor prognosis. We show that TSP1 stimulates a set of processes involved in tumor progression, including cell migration and invasion, extravasation and metastatic dissemination. We also demonstrate that hypoxia induced by an inhibition of angiogenesis resulting from the activity of endogenous or pharmacological molecules is a major activator of invasion and metastasis. In addition, we demonstrate that TSP1 is a poor prognostic factor since its expression is correlated with increased invasion and relapse in patients with androgen-resistant prostate cancer. In conclusion, our results suggest that the expression of TSP1 could be a predictor of invasion and metastasis occurrence, and that targeting TSP1 could be a great therapeutic potential to block these processes., Les métastases représentent l’étape ultime de la progression tumorale. Les traitements inhibiteurs de l’angiogenèse offrent de nouvelles perspectives thérapeutiques, notamment pour les stades invasifs, mais cependant ils n’apportent qu’une prolongation modeste de la survie des patients. La faible réponse de certains types de cancers aux traitements ciblant l’angiogenèse tumorale, comme le carcinome de la prostate chez l’homme ou du sein chez la femme, est due au développement de résistances tumorales d’une part, et à l’accélération du processus métastatique mise en évidence par certaines études précliniques et cliniques d’autre part. L’objectif de ce travail de thèse a été d’étudier le rôle d’une protéine anti-angiogénique endogène, la thrombospondine 1 (TSP1), dans l’invasion et la dissémination métastatique dans le carcinome de la prostate et du sein. Notre laboratoire a montré que la densité microvasculaire est inversement corrélée à l’expression de la TSP1 dans les stades précoces de ces carcinomes. Cependant, cette corrélation est perdue aux stades avancés où l’expression de la TSP1 devient associée à un mauvais pronostic. Les travaux de ma thèse ont permis de montrer que la TSP1 stimule un ensemble de processus intervenant dans la progression tumorale, dont la migration et l’invasion cellulaires, l’extravasation et la dissémination métastatique. Nous avons aussi pu mettre en évidence que l’hypoxie induite par l’inhibition de l’angiogenèse est un activateur majeur de l’invasion et de la formation de métastases, que cette hypoxie soit produite par des molécules endogènes comme la TSP1 ou par des inhibiteurs pharmacologiques de ce processus. De plus, nous démontrons que la TSP1 est un facteur de mauvais pronostic puisque son expression est corrélée avec l’augmentation de l’invasion et de la rechute des patients atteints de cancers de la prostate androgéno-résistants. En conclusion, nos résultats suggèrent que l’expression de la TSP1 pourrait être un facteur prédictif de l’invasion et de l’occurrence de métastases, et que le ciblage de la TSP1 présente un fort potentiel thérapeutique pour bloquer à terme ces processus.