Aleksandra Filipovic

Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer

Therapies targeting estrogen receptor α (ERα, encoded by ESR1) have transformed the treatment of breast cancer. However, large numbers of women relapse, highlighting the need for the discovery of new regulatory targets modulating ERα pathways. An siRNA screen identified kinases whose silencing alters the estrogen response including those previously implicated in regulating ERα activity (such as mitogen-activated protein kinase and AKT). Among the most potent regulators was lemur tyrosine kinase-3 (LMTK3), for which a role has not previously been assigned. In contrast to other modulators of ERα activity, LMTK3 seems to have been subject to Darwinian positive selection, a noteworthy result given the unique susceptibility of humans to ERα+ breast cancer. LMTK3 acts by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), thereby increasing binding of forkhead box O3 (FOXO3) to the ESR1 promoter. LMTK3 phosphorylated ERα, protecting it from proteasomal degradation in vitro. Silencing of LMTK3 reduced tumor volume in an orthotopic mouse model and abrogated proliferation of ERα+ but not ERα− cells, indicative of its role in ERα activity. In human cancers, LMTK3 abundance and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapies. These findings yield insights into the natural history of breast cancer in humans and reveal LMTK3 as a new therapeutic target.

Inhibition of γ-secretase induces G2/M arrest and triggers apoptosis in breast cancer cells

γ-Secretase activity is vital for the transmembrane cleavage of Notch receptors and the subsequent migration of their intracellular domains to the nucleus. Notch overexpression has been associated with breast, colon, cervical and prostate cancers. We tested the effect of three different γ-secretase inhibitors (GSIs) in breast cancer cells. One inhibitor (GSI1) was lethal to breast cancer cell lines at concentrations of 2 μM and above but had a minimal effect on the non-malignant breast lines. GSI1 was also cytotoxic for a wide variety of cancer cell lines in the NCI60 cell screen. GSI1 treatment resulted in a marked decrease in γ-secretase activity and downregulation of the Notch signalling pathway with no effects on expression of the γ-secretase components or ligands. Flow cytometric and western blot analyses indicated that GSI1 induces a G2/M arrest leading to apoptosis, through downregulation of Bcl-2, Bax and Bcl-XL. GSI1 also inhibited proteasome activity. Thus, the γ-secretase inhibitor GSI1 has a complex mode of action to inhibit breast cancer cell survival and may represent a novel therapy in breast cancer.

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

Significance Whether breast cancer will respond to the antiestrogen tamoxifen is determined by whether cellular proliferation is estrogen receptor (ER) α-mediated. As opposed to early ductal cancer, which is an ERα-rich, proliferating disease, in early lobular cancer both ERα and ERβ are abundantly expressed and proliferation is rare. In advanced lobular cancer, ERβ is lost, ERα is retained, and proliferation is high. Thus, tamoxifen may be an effective pharmaceutical in late but not early lobular cancer. The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.

Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo

Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44+/CD24− and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44+/CD24− stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing nicastrin and HCC1806 stably depleted of nicastrin, in vivo, we demonstrate that nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of nicastrin-targeted therapies in breast cancer.

Nicastrin and Notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in MCF7 breast cancer cells

IntroductionResistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.MethodsWe used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen.ResultsWe found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation.ConclusionsETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.

Claudin-1 as a promoter of EMT in hepatocellular carcinoma

Claudins are integral structural and functional components of apical cell adhesions (tight junctions). Loss of such adhesions has been associated with malignant transformation, a process most often accompanied by a concomitant loss of claudin expression. A growing body of evidence reveals the highly contextual upregulation of claudin expression in certain cancer types, and moreover their relevance in promoting cancer cell invasion and metastatic progression. In this issue of Oncogene, Suh et al. reported on claudin-1 expression in hepatocellular carcinoma (HCC), including its role as a promoter of the epithelial−to−mesenchymal transition via the c-Abl/Raf/Ras/ERK signaling pathway. Considering the limited therapeutic options in HCC, evaluation of its role as a target merits further investigation.

LMTK3 is implicated in endocrine resistance via multiple signaling pathways.

Resistance to endocrine therapy in breast cancer is common. With the aim of discovering new molecular targets for breast cancer therapy, we have recently identified LMTK3 as a regulator of the estrogen receptor-alpha (ERα) and wished to understand its role in endocrine resistance. We find that inhibition of LMTK3 in a xenograft tamoxifen (Tam)-resistant (BT474) breast cancer mouse model results in re-sensitization to Tam as demonstrated by a reduction in tumor volume. A whole genome microarray analysis, using a BT474 cell line, reveals genes significantly modulated (positively or negatively) after LMTK3 silencing, including some that are known to be implicated in Tam resistance, notably c-MYC, HSPB8 and SIAH2. We show that LMTK3 is able to increase the levels of HSPB8 at a transcriptional and translational level thereby protecting MCF7 cells from Tam-induced cell death, by reducing autophagy. Finally, high LMTK3 levels at baseline in tumors are predictive for endocrine resistance; therapy does not lead to alteration in levels, whereas in patient’s plasma samples, acquired LMTK3 gene amplification (copy number variation) was associated with relapse while receiving Tam. In aggregate, these data support a role for LMTK3 in both innate (intrinsic) and acquired (adaptive) endocrine resistance in breast cancer.

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

Background:We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown.Methods:A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer.Results:Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53.Conclusion:Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity.

Anti-nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells

The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.

AKT and 14-3-3 Regulate Notch4 Nuclear Localization

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.