Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer.

BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Detection of miRNA regulatory effect on triple negative breast cancer transcriptome.

Identifying key microRNAs (miRNAs) contributing to the genesis and development of a particular disease is a focus of many recent studies. We introduce here a rank-based algorithm to detect miRNA regulatory activity in cancer-derived tissue samples which combines measurements of gene and miRNA expression levels and sequence-based target predictions. The method is designed to detect modest but coordinated changes in the expression of sequence-based predicted target genes. We applied our algorithm to a cohort of 129 tumour and healthy breast tissues and showed its effectiveness in identifying functional miRNAs possibly involved in the disease. These observations have been validated using an independent publicly available breast cancer dataset from The Cancer Genome Atlas. We focused on the triple negative breast cancer subtype to highlight potentially relevant miRNAs in this tumour subtype. For those miRNAs identified as potential regulators, we characterize the function of affected target genes by enrichment analysis. In the two independent datasets, the affected targets are not necessarily the same, but display similar enriched categories, including breast cancer related processes like cell substrate adherens junction, regulation of cell migration, nuclear pore complex and integrin pathway. The R script implementing our method together with the datasets used in the study can be downloaded here (http://bioinfo-out.curie.fr/projects/targetrunningsum).

ß5 integrin is the major contributor to the αVintegrin-mediated blockade of HIV-1 replication.

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. During the differentiation of monocytes to macrophages, adhesion molecules such as integrins are upregulated; therefore, they provide signals that control the process and subsequently may render macrophages more susceptible to HIV-1 infection. Previous work demonstrated that blocking α(v)-containing integrins triggered a signal transduction pathway leading to the inhibition of NF-κB-dependent HIV-1 transcription. In this paper, we show the influence of the different α(v)-coupled ß integrins in HIV-1 replication in macrophages. Inhibition of ß integrins, either by specific mAbs, small arginine-glycine-aspartic acid (RGD) mimetic compounds, or RNA interference, showed that integrin ß(5) was the major contributor to the integrin-mediated blockade of HIV-1 replication. Importantly, such inhibition did not induce changes in cell adhesion to the substrate. In conclusion, our results reveal a significant role of the integrin dimer α(v)ß(5) in HIV-1 infection of macrophages.

Neuropilin-1 is upregulated in hepatocellular carcinoma and contributes to tumour growth and vascular remodelling.

BACKGROUND & AIMS: Neuropilin-1 (NRP1) is a transmembrane co-receptor for semaphorins and heparin-binding pro-angiogenic cytokines, principally members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and malignant progression of many cancers. The role of NRP1 in the development of hepatocellular carcinoma (HCC) is not completely understood. METHODS: We used human tissue microarrays and a mouse transgenic model of HCC to establish the spatio-temporal patterns of NRP1 expression in HCC. To evaluate the therapeutic potential of targeting NRP1 in HCC, we treated HCC mice with peptide N, an NRP1 binding recombinant protein and competitive inhibitor of the VEGF-A(165)/NRP1 interaction. RESULTS: We demonstrate that NRP1 is expressed in hepatic endothelial cells of both human healthy biopsies and in HCC samples, but not in normal hepatocytes. We found that increased NRP1 expression in human tumour hepatocytes is significantly associated with primary HCC. Using RT-PCR, Western blot and immunofluorescence analysis we show that NRP1 expression in the liver of transgenic HCC mice is increased with disease progression, in both vascular and tumour compartments. Blocking NRP1 function with peptide N leads to the inhibition of vascular remodelling and tumour liver growth in HCC mice. CONCLUSIONS: Our results indicate a specific role of NRP1 in HCC growth and vascular remodelling and highlight the possibility of therapeutically targeting NRP1 for the treatment of HCC.

Small interfering RNAs induce target-independent inhibition of tumor growth and vasculature remodeling in a mouse model of hepatocellular carcinoma.

RNA interference mediated by small interfering RNAs (siRNAs) has emerged as a potential therapeutic approach to treat various diseases, including cancer. Recent studies with several animal models of posttraumatic revascularization demonstrated that synthetic siRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3 (TLR3)/interferon pathway and suppression of angiogenesis. To analyze the impact of siRNAs on tumor angiogenesis, we injected transgenic mice developing hepatocellular carcinoma (HCC) with either control siRNAs or siRNA targeting neuropilin-1. We found that treatment with these siRNAs led to a comparable reduction in tumor liver volume and to inhibition of tumor vasculature remodeling. We further determined that TLR3, which recognizes double-stranded siRNA, was up-regulated in mouse HCC. Treatment of HCC mice with polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 agonist, led to both a reduction of tumor liver enlargement and a decrease in hepatic arterial blood flow, indicating that TLR3 is functional and may mediate both anti-angiogenic and anti-tumor responses. We also demonstrated that siRNAs increased interferon-γ levels in the liver. In vitro, interferon-γ inhibited proliferation of endothelial cells. In addition, we found that siRNAs inhibited endothelial cell proliferation and morphogenesis in an interferon-γ-independent manner. Our results suggest that synthetic siRNAs inhibit target-independently HCC growth and angiogenesis through the activation of the innate interferon response and by directly inhibiting endothelial cell function.

Cell adhesion through alphaV-containing integrins is required for efficient HIV-1 infection in macrophages.

Monocytes and macrophages are an important reservoir of human immunodeficiency virus (HIV) and may represent the largest reservoir of this virus in tissues. Differentiation of monocytes into macrophages leads to cell attachment and susceptibility to infection and replication of HIV. Among other cell-surface molecules, integrins are overexpressed during monocyte-macrophage differentiation and may play a role in the replication cycle of envelope viruses including HIV. Here, we show that inhibition of alphaV integrin in monocyte-derived macrophages, by RNA interference or their inhibition by a selective small heterocyclic RGD-mimetic nonpeptide compound, inhibited the replication of HIV in the absence of cytotoxicity. Interference or inhibition of alphaV integrins triggered a signal transduction pathway, leading to down-regulation of nuclear factor-kappaB-dependent HIV-1 transcription. Such inhibition was mediated by a MAP-kinase signaling cascade, probably involving ERK1/2, p38-mitogen-activated protein kinases, and HSP27. In conclusion, our results reveal a significant role of integrin alphaV-mediated adhesion in HIV-1 infection of macrophages.

Capparis kbangensis (Capparaceae), a new species from central Vietnam.

Capparis kbangensis Sy & D.V. Hai, a new species from Kbang District, Gia Lai Province, Vietnam, is described and illustrated. The new species is morphologically similar to Capparis versicolor but differs by several characters such as emarginate leaf apex, hairy margin of sepals, smaller fruits, and fewer seeds per fruit. Its ecology and conservation status are provided along with a taxonomic key to the closely allied species.

LRP8 is overexpressed in estrogen-negative breast cancers and a potential target for these tumors.

Triple-negative breast cancer (TNBC) is the breast cancer subtype with the worst prognosis. New treatments improving the survival of TNBC patients are, therefore, urgently required. We performed a transcriptome microarray analysis to identify new treatment targets for TNBC. We found that low-density lipoprotein receptor-related protein 8 (LRP8) was more strongly expressed in estrogen receptor-negative breast tumors, including TNBCs and those overexpressing HER2, than in luminal breast tumors and normal breast tissues. LRP8 depletion decreased cell proliferation more efficiently in estrogen receptor-negative breast cancer cell lines: TNBC and HER2 overexpressing cell lines. We next focused on TNBC cells for which targeted therapies are not available. LRP8 depletion induced an arrest of the cell cycle progression in G1 phase and programmed cell death. We also found that LRP8 is required for anchorage-independent growth in vitro, and that its depletion in vivo slowed tumor growth in a xenograft model. Our findings suggest that new approaches targeting LRP8 may constitute promising treatments for hormone-negative breast cancers, those overexpressing HER2 and TNBCs.

Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells.

INTRODUCTION: Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs. METHODS: In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines. RESULTS: The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition. CONCLUSIONS: These data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.

S32826, a nanomolar inhibitor of autotaxin: discovery, synthesis and applications as a pharmacological tool.

Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [(14)C]lyso-phosphatidylcholine conversion into [(14)C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC(50) in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.

LRP5 regulates the expression of STK40, a new potential target in triple-negative breast cancers.

Triple-negative breast cancers (TNBCs) account for a large proportion of breast cancer deaths, due to the high rate of recurrence from residual, resistant tumor cells. New treatments are needed, to bypass chemoresistance and improve survival. The WNT pathway, which is activated in TNBCs, has been identified as an attractive pathway for treatment targeting. We analyzed expression of the WNT coreceptors LRP5 and LRP6 in human breast cancer samples. As previously described, LRP6 was overexpressed in TNBCs. However, we also showed, for the first time, that LRP5 was overexpressed in TNBCs too. The knockdown of LRP5 or LRP6 decreased tumorigenesis in vitro and in vivo, identifying both receptors as potential treatment targets in TNBC. The apoptotic effect of LRP5 knockdown was more robust than that of LRP6 depletion. We analyzed and compared the transcriptomes of cells depleted of LRP5 or LRP6, to identify genes specifically deregulated by LRP5 potentially implicated in cell death. We identified serine/threonine kinase 40 (STK40) as one of two genes specifically downregulated soon after LRP5 depletion. STK40 was found to be overexpressed in TNBCs, relative to other breast cancer subtypes, and in various other tumor types. STK40 depletion decreased cell viability and colony formation, and induced the apoptosis of TNBC cells. In addition, STK40 knockdown impaired growth in an anchorage-independent manner in vitro and slowed tumor growth in vivo. These findings identify the largely uncharacterized putative protein kinase STK40 as a novel candidate treatment target for TNBC.

Inhibitors of integrins.

The inhibition of integrins--cell surface receptors with a crucial role in angiogenesis, tumour cell survival, invasion and metastases--has centred on the alpha(v)beta3 integrin. Work has culminated in two antagonists that are in clinical trials as cancer therapeutics. Other integrins appear to be candidate targets in the light of gene knockout studies. Surprisingly, genetic alpha(v)beta3 ablation did not confirm the pertinence of the use of alpha(v)beta3 antagonists. However, these apparent discrepancies could be explained by the new finding that this integrin has a role as a cell survival sensor, limiting rather than promoting angiogenesis. Accumulating data on the role of integrins and the mechanism of action of pharmacological antagonists will help to develop and apply an efficient anti-integrin therapy in cancer.

Transcriptome analysis of Wnt3a-treated triple-negative breast cancer cells.

The canonical Wnt/ß-catenin pathway is activated in triple-negative breast cancer (TNBC). The activation of this pathway leads to the expression of specific target genes depending on the cell/tissue context. Here, we analyzed the transcriptome of two different TNBC cell lines to define a comprehensive list of Wnt target genes. The treatment of cells with Wnt3a for 6h up-regulated the expression (fold change > 1.3) of 59 genes in MDA-MB-468 cells and 241 genes in HCC38 cells. Thirty genes were common to both cell lines. Beta-catenin may also be a transcriptional repressor and we found that 18 and 166 genes were down-regulated in response to Wnt3a treatment for 6h in MDA-MB-468 and HCC38 cells, respectively, of which six were common to both cell lines. Only half of the activated and the repressed transcripts have been previously described as Wnt target genes. Therefore, our study reveals 137 novel genes that may be positively regulated by Wnt3a and 104 novel genes that may be negatively regulated by Wnt3a. These genes are involved in the Wnt pathway itself, and also in TGFß, p53 and Hedgehog pathways. Thorough characterization of these novel potential Wnt target genes may reveal new regulators of the canonical Wnt pathway. The comparison of our list of Wnt target genes with those published in other cellular contexts confirms the notion that Wnt target genes are tissue-, cell line- and treatment-specific. Genes up-regulated in Wnt3a-stimulated cell lines were more strongly expressed in TNBC than in luminal A breast cancer samples. These genes were also overexpressed, but to a much lesser extent, in HER2+ and luminal B tumors. We identified 72 Wnt target genes higher expressed in TNBCs (17 with a fold change >1.3) which may reflect the chronic activation of the canonical Wnt pathway that occurs in TNBC tumors.

TIPIN depletion leads to apoptosis in breast cancer cells.

Triple-negative breast cancer (TNBC) is the breast cancer subgroup with the most aggressive clinical behavior. Alternatives to conventional chemotherapy are required to improve the survival of TNBC patients. Gene-expression analyses for different breast cancer subtypes revealed significant overexpression of the Timeless-interacting protein (TIPIN), which is involved in the stability of DNA replication forks, in the highly proliferative associated TNBC samples. Immunohistochemistry analysis showed higher expression of TIPIN in the most proliferative and aggressive breast cancer subtypes including TNBC, and no TIPIN expression in healthy breast tissues. The depletion of TIPIN by RNA interference impairs the proliferation of both human breast cancer and non-tumorigenic cell lines. However, this effect may be specifically associated with apoptosis in breast cancer cells. TIPIN silencing results in higher levels of single-stranded DNA (ssDNA), indicative of replicative stress (RS), in TNBC compared to non-tumorigenic cells. Upon TIPIN depletion, the speed of DNA replication fork was significantly decreased in all BC cells. However, TIPIN-depleted TNBC cells are unable to fire additional replication origins in response to RS and therefore undergo apoptosis. TIPIN knockdown in TNBC cells decreases tumorigenicity in vitro and delays tumor growth in vivo. Our findings suggest that TIPIN is important for the maintenance of DNA replication and represents a potential treatment target for the worst prognosis associated breast cancers, such as TNBC.

Stereospecific synthesis of 5-substituted 2-bisarylthiocyclopentane carboxylic acids as specific matrix metalloproteinase inhibitors.

The synthesis and structure-activity relationship (SAR) studies of a series of cyclopentane carboxylic acid matrix metalloproteinase (MMP) inhibitors are described. Potent and specific MMP-2, -3, -9, -13 inhibitors were obtained by regio- and stereoselective substitutions at positions 2 and 5 on the cyclopentane ring. Compounds 2a and 2e are active in the mouse B16-F10 metastasis model and display very good pharmacokinetic parameters.

Alpha v integrin inhibitors and cancer therapy.

Recent gene disruption experiments have suggested that targeting the alpha v integrins (cell surface adhesion and signaling receptors) to prevent tumor progression can result in different outcomes depending on the strategy. Nevertheless, two alpha v binding antagonists have made their way to the clinic in the oncology field; both Vitaxin, a humanized antibody, and cilengitide, a cyclic peptide mimicking the RGD ligand recognition peptidic domain common to alpha v integrin ligands, are in phase II clinical trials. This year, development of another peptidic inhibitor was initiated. This review questions whether companies are reluctant to propose small synthetic heterocyclic inhibitors as successors to peptide-derived agents with better pharmacokinetics and oral bioavailability. Is this class of compounds immediably flawed like the platelet alpha IIb beta 3 oral antagonists? Tentative answers are provided in this review following description of the lead compounds and the rationale for their use as cancer treatments, imaging agents or drug targeting vectors.

TTK/hMPS1 is an attractive therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers (BC) associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment of patients with TNBC. In order to discover potential therapeutic targets, we searched for protein kinases that are overexpressed in human TNBC biopsies and whose silencing in TNBC cell lines causes cell death. A cohort including human BC biopsies obtained at Institut Curie as well as normal tissues has been analyzed at a gene-expression level. The data revealed that the human protein kinase monopolar spindle 1 (hMPS1), also known as TTK and involved in mitotic checkpoint, is specifically overexpressed in TNBC, compared to the other BC subgroups and healthy tissues. We confirmed by immunohistochemistry and reverse phase protein array that TNBC expressed higher levels of TTK protein compared to the other BC subgroups. We then determined the biological effects of TTK depletion by RNA interference, through analyses of tumorigenic capacity and cell viability in different human TNBC cell lines. We found that RNAi-mediated depletion of TTK in various TNBC cell lines severely compromised their viability and their ability to form colonies in an anchorage-independent manner. Moreover, we observed that TTK silencing led to an increase in H2AX phosphorylation, activation of caspases 3/7, sub-G1 cell population accumulation and high annexin V staining, as well as to a decrease in G1 phase cell population and an increased aneuploidy. Altogether, these data indicate that TTK depletion in TNBC cells induces apoptosis. These results point out TTK as a protein kinase overexpressed in TNBC that may represent an attractive therapeutic target specifically for this poor prognosis associated subgroup of breast cancer.

Polo-like kinase 1: a potential therapeutic option in combination with conventional chemotherapy for the management of patients with triple-negative breast cancer.

Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. To discover potential therapeutic targets for the poor prognosis-associated triple-negative breast cancer (TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1 (PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes. High PLK1 expression was confirmed by reverse phase protein and tissue microarrays. In triple-negative cell lines, RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increase in phosphorylated H2AX, G(2)-M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencing impaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel), and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line. When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo in two xenografts models established from biopsies of patients with TNBC. Most importantly, the administration of BI-2536, in combination with doxorubicin + cyclophosphamide chemotherapy, led to a faster complete response compared with the chemotherapy treatment alone and prevented relapse, which is the major risk associated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeutic approach, in association with conventional chemotherapy, for the management of patients with TNBC.

alphavbeta3 Integrin-targeting Arg-Gly-Asp (RGD) peptidomimetics containing oligoethylene glycol (OEG) spacers.

RGD peptides are used in biomaterials science for surface modifications with a view to elicit selective cellular responses. Our objective is to replace peptides by small peptidomimetics acting similarly. We designed novel molecules targeting alpha(v)beta(3) integrin and featuring spacer-arms (for surface grafting), which do not disturb the biological activity, from (l) N-(3-(trifluoromethyl)benzenesulfonyl) tyrosine used as scaffold. Various Arg-mimics were fixed on the phenol function, and the ortho position was used for the coupling of OEG spacers. All peptidomimetics were active in the nM range in a binding test toward human alpha(v)beta(3) integrin (IC(50) = 0.1 to 1.7 nM) and selective versus platelet integrin alpha(IIb)beta(3). Selected compounds revealed excellent ability to inhibit bone cells adhesion on vitronectin. Modeling and docking studies were performed for comparing the most active RGD peptidomimetic to cilengitide, i.e., cyclo-[RGDfN(Me)V]-. Lastly, the adhesion of endothelial cells on a cultivation support grafted with RGD peptidomimetics was significantly improved.

Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors.

Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.