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 þ 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. Cancer Res; 73(2); 813-23. Ó2012 AACR.
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.
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.
Background: Triple-negative breast cancer (TNBC) is associated with poor prognosis, only partial response to chemotherapy and lack of clinically established targeted therapies [1]. A deregulation of the Wnt signaling pathway has been described in breast cancers, particularly in TNBC [2–6]. Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical beta-catenin pathway. An overexpression of LRP6 has been reported to enhance Wnt signaling favoring in vitro cell proliferation and in vivo mammary gland hyperplasia and tumor growth [5,7,8]. LRP6 has been claimed to be a potential TNBC therapeutic target [5]. Material and Methods: RNA microarray analysis and reverse phase protein array were performed on 154 samples including biopsies of the various subclasses of breast cancer. MDA-MB-468 and HCC38 cell lines were defined as the most representative in vitro models of the Wnt pathway status found in TNBC biopsies. In order to study the functions of LRP5 or LRP6 in TNBC, we examined in these cell lines the effects of their depletions using RNAi technology on tumorigenesis and on the Wnt3a-induced signaling pathway. Results: Our transcriptomic and proteomic data revealed that both LRP5 and LRP6 are overexpressed in TNBC compared to the other breast cancer subtypes i.e. HER2+/ER-, luminal A and luminal B. Our in vitro studies indicated that the transcriptional activity of beta-catenin/Tcf was strongly reduced when LRP6 was silenced and to a lesser extend when LRP5 was depleted. In accordance with these results, the expression of AXIN2 and other newly identified Wnt target genes, was mainly down-regulated in cells silenced for LRP6. LRP5 and LRP6 knockdown impaired colony formation in soft agar and weakly decreased the number of mammospheres. The inhibition of cell viability observed after LRP5 depletion was the consequence of a programmed cell death as revealed by the increase of annexin V-positive cells, the activation of initiator and effector caspases (8,9,3/7) and the cleavage of poly(ADP-ribose) polymerase. On the contrary, LRP6 depletion inhibited cell viability without promoting apoptosis as reported by others [5]. Conclusions: Altogether our data demonstrate that in TNBC cell lines, LRP5 or LRP6 silencing has an impact on Wnt signaling, cancer stem cell-like activity, tumorigenic properties and cell viability. Most importantly, LRP5 silencing promotes apoptosis, suggesting that LRP5 could represent a promising therapeutic candidate to target in TNBC. Citation Format: Sylvie Maubant, Virginie Maire, Bruno Tesson, David Gentien, Bérengère Marty-Prouvost, Francisco Cruzalegui, Stéphane Depil, Gordon C. Tucker, Sergio Roman-Roman, Thierry Dubois. LRP5: a potential therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4373. doi:10.1158/1538-7445.AM2013-4373
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 Némati, Aurélie Dumont, David Gentien, Bérengère Marty-Prouvost, Guillem Rigaill, Leanne De Koning, Anne Vincent-Salomon, Emmanuel Barillot, Didier Decaudin, Alain Pierré, Stéphane 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.
Results confirmed the reliability and the utility of RPPA for screening global changes induced in signaling pathways by kinase inhibitors. MAPK and AKT were identified as the main pathways involved in vandetanib response in MTC models. Our results also suggest alternative routes for controlling the disease and provide a rationale for the development of therapeutic combinations based on the comprehensive identification of molecular events induced by inhibitors.
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 Némati, David Gentien, Bérengère Marty-Prouvost, Stéphane 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
Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. Our aim is to discover potential therapeutic targets for triple negative breast cancer (TNBC), a poor prognosis subgroup with no targeted therapy available yet. Gene expression profiling on a cohort of 160 breast cancers including 40 TNBC revealed that the polo-like kinase 1 (PLK1), a protein kinase, is specifically over-expressed in TNBC compared to the other breast cancer subtypes. High PLK1 expression was confirmed at a proteomic level by reverse phase protein array and tissue microarray. In TNBC cell lines grown as monolayers, RNAi-mediated PLK1 depletion or small compound (BI-2536)-mediated PLK1 inhibition induced an increase in phosphorylated H2AX, G2/M arrest and apoptosis, resulting in a decrease in cell viability. In addition, a soft-agar colony assay showed that PLK1 silencing impaired tumorigenicity of TNBC cell lines. With cells grown in extracellular matrix gels (Matrigel), BI-2536 induced apoptosis specifically in TNBC cancerous versus “normal” cell lines. The in vivo anti-tumor effect of BI-2536 was investigated in two TNBC xenografts derived from patient's biopsies. When administrated as a single agent, the PLK1 inhibitor significantly impaired the tumor growth. Most importantly, administration of BI-2536 in combination with adriamycin + cyclophosphamide chemotherapy led to 100% complete response (9/9 mice). Altogether, our observations point out that PLK1 may represent an attractive therapeutic target, in association with conventional chemotherapy, for the management of patients with TNBC. 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 2938. doi:1538-7445.AM2012-2938
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