Francisco Cruzalegui

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

IntroductionBasal-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.MethodsIn 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.ResultsThe 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.ConclusionsThese 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.

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.

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.

S49076 is a Novel Kinase Inhibitor of MET, AXL and FGFR with Strong Preclinical Activity Alone and in Association with Bevacizumab

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749–62. ©2013 AACR.

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

The canonical Wnt/β-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 TGFβ, 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.

Structure-Based Design and Synthesis of Harmine Derivatives with Different Selectivity Profiles in Kinase versus Monoamine Oxidase Inhibition

Dual‐specificity tyrosine‐phosphorylation‐regulated kinase 1A (DYRK1A) is an emerging biological target with implications in diverse therapeutic areas such as neurological disorders (Down syndrome, in particular), metabolism, and oncology. Harmine, a natural product that selectively inhibits DYRK1A amongst kinases, could serve as a tool compound to better understand the biological processes that arise from DYRK1A inhibition. On the other hand, harmine is also a potent inhibitor of monoamine oxidase A (MAO‐A). Using structure‐based design, we synthesized a collection of harmine analogues with tunable selectivity toward these two enzymes. Modifications at the 7‐position typically decreased affinity for DYRK1A, whereas substitution at the 9‐position had a similar effect on MAO‐A inhibition but DYRK1A inhibition was maintained. The resulting collection of compounds can help to understand the biological role of DYRK1A and also to assess the interference in the biological effect originating in MAO‐A inhibition.

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.

Abstract 2764: The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target

Treatment of patients with triple-negative breast cancers (TNBCs) remains a major challenge for oncologists and alternative treatments to conventional chemotherapies are needed to improve their survival. The Wnt/beta-catenin signaling, recently reported to be activated in TNBCs, may represent an interesting pathway to target. We report that both LRP5 and LRP6 Wnt coreceptors are more strongly expressed in TNBCs than in other breast tumor subtypes. As very few studies have explored potential differences between LRP5 and LRP6, we investigated the effects of modulating specifically LRP5 or LRP6 expression on Wnt signaling, cell viability and tumorigenesis in HCC38 and MDA-MB-468 TNBC cells. We found that these two cell lines are more similar to TNBC biopsy specimens in terms of Wnt pathway gene expression profiles than any other tested cell line. Unlike LRP5, LRP6 was involved in activating the canonical Wnt pathway in response to Wnt3a. LRP5 knockdown induced caspase-dependent apoptosis, whereas LRP6 knockdown had no such effect. Importantly, LRP5-depleted cells were more sensitive to conventional chemotherapy than cells depleted of LRP6. The knockdown of LRP5 or LRP6 decreased tumorigenesis both in vitro and in vivo. Overall, these data suggest that the LRP5 and LRP6 coreceptors have different functions in TNBCs, with LRP5 playing a preponderant role in survival control. Our data suggest that both LRP5 and LRP6 are potential treatment targets in TNBCs, but that LRP5 may be the most useful target, given the impact of its depletion on cell survival as well as on the response to anti-cancer drugs. Citation Format: Sylvie Maubant, Virginie Maire, Bruno Tesson, Fariba Nemati, David Gentien, Berengere Marty-Prouvost, Stephane Depil, Francisco Cruzalegui, Gordon Tucker, Sergio Roman-Roman, Thierry Dubois. The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2764. doi:10.1158/1538-7445.AM2014-2764

Abstract B233: The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target.

Introduction. Treatment of patients with triple-negative breast cancers (TNBCs) remains a major challenge for oncologists. Although they respond well to the current therapeutic strategies based on conventional chemotherapies, they represent a large proportion of breast cancer death due to a high recurrence rate. Alternative treatments are needed to improve survival of these patients. The Wnt/beta-catenin signaling, recently reported to be activated in TNBCs, may represent an interesting pathway to target. Methods. We analyzed mRNA, DNA and protein levels for the LRP5 and LRP6 Wnt coreceptors in our cohort of breast tumor biopsy specimens. We then identified which TNBC cell lines display the most similarity to TNBC tumors regarding the Wnt pathway status using a centroid approach. We investigated the effects of modulating LRP5 or LRP6 expression on Wnt signaling, cell viability and apoptosis. We evaluated the potential therapeutic value of targeting LRP5 and LRP6 in TNBCs, by performing depletion experiments and treating cells with a mixture of doxorubicin/cyclophosphamide. We also examined whether the depletion of LRP5 or LRP6 had an impact on tumorigenicicy in vitro, in soft-agar assays, and in vivo, in xenograft models. Results. Gene expression analyses revealed that both LRP5 and LRP6 Wnt coreceptors were more strongly expressed in TNBCs than in other breast tumor subtypes. HCC38 and MDA-MB-468 TNBC cells were more similar to TNBC biopsy specimens in terms of Wnt pathway gene expression profiles than any other tested cell line. Unlike LRP5, LRP6 was involved in activating the canonical Wnt pathway in response to Wnt3a. LRP5 knockdown induced caspase-dependent apoptosis, whereas LRP6 knockdown had no such effect. LRP5-depleted cells were also more sensitive to conventional chemotherapy than cells depleted of LRP6. The knockdown of LRP5 or LRP6 decreased tumorigenesis both in vitro and in vivo. Conclusions. These data indicate that the LRP5 and LRP6 have different functions in TNBCs, with LRP5 playing a preponderant role in survival control. Our data suggest that both coreceptors are potential treatment targets in TNBCs, but that LRP5 may be the most useful target, given the impact of its depletion on cell survival and the response to anti-cancer drugs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B233. Citation Format: Sylvie Maubant, Virginie Maire, Bruno Tesson, Fariba Nemati, Aurelie Dumont, David Gentien, Berengere Marty-Prouvost, Guillem Rigaill, Leanne De Koning, Anne Vincent-Salomon, Emmanuel Barillot, Didier Decaudin, Alain Pierre, Stephane Depil, Francisco Cruzalegui, Gordon C. Tucker, Sergio Roman-Roman, Thierry Dubois. The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B233.

Abstract 3000: Molecular profiling of residual tumor cells after various chemotherapies shows distinct gene expression patterns in patient-derived breast cancer xenografts

Introduction: Triple negative breast cancer (TNBC) is an aggressive disease associated with a high risk of distant recurrence, rapid progression to death and poor overall survival. Systemic treatments of TNBC patients are limited to cytotoxic chemotherapy due to the lack of identified molecular targets. Therefore, the characterization of tumor cells that are responsible for recurrences after such treatments is of high therapeutic relevance. Using patient-derived TNBC xenografts that reproduce tumor relapse, we have analyzed the gene expression profile of residual cancer cells (RCC) after various chemotherapeutic treatments. Experimental design: Sixteen human breast cancer xenografts (HBCx) were treated with adriamycin-cyclophosphamide (AC), docetaxel, cisplatin, or capecitabine. Xenografts showing complete tumor remission followed by local relapse in at least 75% of mice were considered as tumor recurrence models. Gene expression profiles were obtained from tumor samples before treatment (untreated group) and after treatment (tumor remission and tumor recurrence groups) using Affymetrix U133 Gene Arrays. Results: Two xenograft models showed complete tumor remission after chemotherapy, i.e. the HBCx-10 xenograft (AC) and the HBCx-33 xenograft (cisplatin and capecitabine). An exploratory analysis on expression datasets of these xenografts tumors showed two distinct groups: one consisted of untreated and relapsed tumors and the other of RCC. The differential analysis showed no difference in gene expression between the untreated and relapsed tumors in the 2 models analyzed, whatever the cytotoxic agent administered. In contrast, when gene expression was analyzed in RCC compared to untreated or relapsed tumors, we found 2405 genes differentially expressed in the HBCx-10 xenograft (fold change greater than 2 or less than −2), and 2886 and 945 genes in the HBCx-33 one treated with capecitabine and cisplatin, respectively. By comparing the 3 lists of differentially expressed genes, we found 227 common genes, 137 being up-regulated and 90 down-regulated. Gene Ontology analysis of these genes revealed enrichment in several biological processes related to immune response, response to wounding, inflammatory response, cell proliferation, cell adhesion, and apoptosis. Validation studies with additional RNA and protein studies are currently ongoing on selected deregulated genes. Conclusions This study supports the existence of a distinct molecular profile of residual tumor cells after chemotherapy in TNBC, with a subset of deregulated genes common to the different tumors analyzed and the different administered treatments. Further elucidation of the signaling pathways associated to these deregulated genes may provide key insights into the molecular mechanism driving breast cancer recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3000. doi:1538-7445.AM2012-3000