Luminal Breast Cancer Models Research Articles

Abstract P6-04-14: Targeting the PI3K/mTOR pathway in patient-derived xenograft models of endocrine resistant luminal breast cancer

Abstract Background. Most ER+ tumors will eventually escape endocrine treatments. PI3K/mTOR pathway targeting has improved clinical results in endocrine treatments resistant (ETR) patients, however with unsustained responses. Our laboratory has developed several models of ETR luminal breast cancer (LBC) patient derived xenografts (PDX) suitable for biological studies (Cottu et al, AACR 2012 & IMPAKT 2012). The aims of the present study were 1) to analyze the biological factors associated with acquired ETR in relevant models of LBC; 2) to determine the therapeutic interest of combining everolimus with different ET models resistant to different endocrine treatments (ET). Methods. Implementation of LBC models has been reported (Cottu et al, BCRT 2012). ETR models were derived by selecting tumors growing under continuous ET, subsequently re-engrafted and rechallenged with endocrine therapies (tamoxifen, letrozole, fulvestrant), and with everolimus alone and combined with ETs. When possible, initial sensitive tumors were simultaneously treated with the same protocols. At progression, or after at least 120 days of continuous therapies, tumor tissue was retrieved. Tissue microarrays (TMA) were performed to analyze the biological modifications associated to ET and mTOR targeting. Triplicate immunohistochemistry of P-AKT/AKT, P-mTOR/mTOR and P-S6 were performed in addition to standard markers. Transcriptomic and RTPCR analyses are also being conducted. Results. Three models resistant to tamoxifen and 2 models resistant to surgical ovariectomy used a surrogate to estrogen deprivation, were developed from 3 initial PDX, and were maintained as independent hormone-resistant variants. All these models retained a luminal phenotype, based on IHC and transcriptomic analyses and had a wt PI3KCA. ETR models had an expression profile very similar to the initial PDX, suggesting that minor changes may be associated with the acquisition of an ETR phenotype. Three of these models have been tested. One tamoxifen resistant model confirmed a resistance specific to tamoxifen only and a high level of sensitivity to a fulvestrant everolimus combination. A second PDX yielded 2 other models resistant to tamoxifen and ovariectomy. These 2 models developed a resistance to all ET modalities. Therapeutic testing with combinations of everolimus and ETs were highly efficient, but everolimus alone was as efficient in these 2 models with complete and durable responses. Activation of the PI3K pathway was demonstrated in all cases by IHC analyses (expression of pAKT and pS6), and complementary ongoing RTPCR analyses. Everolimus based effects were associated with tumor fibrosis, a strong inhibition of Ki67 and pS6, but not always pAKT. The expression of ER was not modified by treatments, except in the fulvestrant-treated mice where it was strongly inhibited. Conclusion. We have developed and validated a platform of ETR models derived from our LBC original PDX. These models retained a luminal phenotype although highly resistant to ET. Activation of the PI3K pathway has been confirmed in ETR models, and everolimus alone, or combined with ET confirms high and durable efficacy. Updated mechanistic analyses performed at different time points will be presented at the meeting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-14.

Abstract P1-05-02: Epithelial-to-epithelial transition precedes collective breast cancer invasion

Abstract BACKGROUND: Invasion represents a fundamental step in tumor progression and is a driving force for metastasis and cancer-related mortality. Invasion has been studied largely in the context of single cells, but in vivo, most tumor cells are not found in isolation, but rather are found as ensembles of cells with substantial molecular heterogeneity. It remains poorly understood in this context which cells are invasive, how these cells arise or how these cells interact collectively to accomplish invasion. RESULTS: We developed an ex-vivo 3D culture assay enabling us to distinguish invasive from non-invasive cells and to interrogate their molecular phenotype. In two mouse models of luminal breast cancer, we find that invasive cells express markers of basal epithelium including cytokeratin K14, p63 and P-cadherin. Invasive K14+ cells were also molecularly and functionally distinct from normal myoepithelial cells and co-expressed luminal K8 and E-cadherin. In vivo, K14+ cells were localized to tumor-stromal interfaces in the primary tumor and in lung micro and macro-metastases. Using a molecular biosensor and time-lapse microscopy, we observed that K14+ invasive cells were derived from K14-negative luminal tumor cells by direct phenotypic conversion. Interestingly, the capacity of luminal tumor cells to acquire basal markers varied by model, with few K14+ cells in MMTV-Her2 and many K14+ cells in MMTV-PyMT mice, correlating with the known in vivo kinetics of tumor progression in these models. To block this epithelial to epithelial transition, we tested the activity of an inhibitor of ribosome S6 kinase (RSK), which has been shown previously to inhibit myoepithelial differentiation in normal mammary epithelium. RSK inhibition caused relative depletion of K14+ tumor cells in 3D culture and abrogation of invasion. Finally, our assay revealed that in fresh primary tissue from human ER+ luminal breast cancers, invasive cells also expressed basal epithelial markers. CONCLUSIONS: Taken together, these data suggest that an epithelial-to-epithelial transition precedes collective invasion in luminal breast cancers. Because we observe K14+ leader cells independent of tumor estrogen receptor status, these data may be generally applicable to breast cancer invasion across subtypes. Because many epithelial tissues have basal populations, K14+ tumor cells may also lead invasion in other cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-02.

IGF1 dependence of dietary energy balance effects on murine Met1 mammary tumor progression, epithelial-to-mesenchymal transition, and chemokine expression

Luminal breast tumors with little or no estrogen receptor α expression confer poor prognosis. Using the Met1 murine model of luminal breast cancer, we characterized the IGF1-dependency of diet-induced obesity (DIO) and calorie restriction (CR) effects on tumor growth, growth factor signaling, epithelial-to-mesenchymal transition (EMT), and chemokine expression. Liver-specific IGF1-deficient (LID) and littermate control (LC) mice were administered control, DIO, or 30% CR diets for 3 months before orthotopic injection of Met1 cells. Tumors grew for 1 month and then were assessed for Akt pathway activation and mRNA expression of chemokine and EMT constituents. LID mice, regardless of diet, displayed reduced Met1 tumor growth and downregulated Akt, EMT, and chemokine pathways. CR, relative to control, reduced serum IGF1 and Met1 tumor growth in LC (but not LID) mice. DIO, relative to control, increased Met1 tumor growth and chemokine expression in LID mice, and had no effect on serum IGF1 or pAkt or cyclin D1 expression in either genotype. Thus, circulating IGF1 (in association with Akt, EMT, and chemokines) regulated Met1 tumor growth. While the anticancer effects of CR were largely IGF1-dependent, the procancer effects of DIO manifested only when circulating IGF1 levels were low. Thus, in a murine model of luminal breast cancer, IGF1 and its downstream signaling pathway, EMT, and chemokines present possible mechanistic regulatory targets. Transplanted MMTV1 Wnt1 mammary tumor growth was also reduced in LID mice, relative to LC mice, suggesting that the IGF1 effects on mammary tumor growth are not limited to Met1 tumors.

Abstract SY02-02: Novel mechanisms regulating breast cancer progression, metastasis, and response to therapy

Abstract In 1863, Virchow hypothesized that the cancer originated at sites of chronic tissue injury and that the ensuing inflammation they cause enhances cell proliferation. While it is now clear that proliferation of cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma and DNA damage promoting agents, certainly potentiates and/or promotes neoplastic risk. In fact, in some cases, the trigger for neoplastic progression may even come from signals within the stromal microenvironment. The stromal microenvironment consists of several cell types (fibroblasts, macrophages, vascular components, and inflammatory cells of the innate and acquired immune response), as well as the extracellular matrix that they elaborate and all the molecules that are concentrated and immobilized on it. All of these components communicate with each other and with the neoplastic cells to contribute to the aberrant tumor organ. We used genetic and imaging techniques to study the interaction between inflammatory and epithelial cancer cells during tumor progression. We visualized the behavior of the cancer cells and leukocytes in living, anaesthetized mice using a spinning disk confocal microscope. We found that leukocytes observed at the tumor-stroma interface are very motile whereas those within the tumor are relatively immotile. The inflammatory switch occurred in parallel with the malignant conversion of the tumor cells. Transcriptional regulation of epithelial differentiation, cell-cell interaction and motility was a key intrinsic event in tumor progression. Tumor cell progression was regulated by GATA3, a master regulator for luminal differentiation, which was lost upon malignant progression, when the tumor cells disseminated throughout the body. Restoration of GATA3 into the malignant cells prevented the earliest stage of tumor metastasis, tumor cell dissemination, and altered the stromal response including decreasing angiogenesis. Zeppo1, a transcriptional repressor present in an amplicon on human chromosome 8p12, regulated epithelial cell adhesion and migration, enhancing later events in metastasis. The inflammatory cells modulate the tumor ecology, altering vascular permeability and responses to chemotherapy. The dynamic interplay of tumor cells and host cells responding to the tumor contribute to tumor evolution and evasion of therapeutic responses.Our data support the hypothesis that that both extrinsic and intrinsic mechanisms regulate tumor progression. Egeblad, M., A. J. Ewald, et al. (2008). Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis. Model. Mech. 1:155-167. PMID: 1904807 Ewald, A.J., A. Brenot, M. Duong, B.S. Chan & Z. Werb (2008). Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell. 14:570-581. PMID: 18410732. Kouros-Mehr, H., S. K. Bechis, et al. (2008). GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. Cancer Cell. 13:141-52. PMID: 18242514. Lu, P. & Z. Werb (2008). Patterning mechanisms of branched organs. Science. 322:1506-1509. PMID: 19056977. Welm, B.E, G. J. P. Dijkgraaf, A. S. Bledau, A. L. Welm & Z. Werb (2008). Lentiviral transduction of stem cells for genetic analysis of mammary development and breast cancer. Cell Stem Cell. 2:90-102. PMID: 18371425. Citation Format: Zena Werb. Novel mechanisms regulating breast cancer progression, metastasis, and response to therapy [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY02-02

Abstract A17: Transforming growth factor beta stimulates STAT3 tyrosine phosphorylation and cellular invasion through interleukin-6 in human breast cancer cell lines

Abstract The signaling mechanisms involving Transforming Growth Factor β (TGFβ) and Signal Transducer and Activator of Transcription 3 (STAT3) are characterized in breast cancer, but the mechanism of crosstalk between these two pathways is poorly understood. In this study we addressed the question of TGFβ-STAT3 cross-signaling in nontransformed epithelial cells and in human breast cancer cell lines. Breast cancer continues to be a serious disease in the United States. A large component of novel chemotherapeutics specifically target overexpressed or overactivated proteins in cancers. Two examples of these oncogenic molecules are the cytokine TGFβ and the transcription factor STAT3. Although these proteins have each been implicated in early and late stages of cancer and invasion, the mechanism of cross-talk between the TGFβ and STAT3 pathways has not been well characterized. We have observed that TGFβ treatment is sufficient to induce phosphorylation of STAT3 on its activating Tyr (705) site in nontransformed mouse mammary NMuMG epithelial cells. The time dependence of this relationship implies de novo expression of an intermediate factor that data suggest is Interleukin-6 (IL-6). TGFβ treatment induces gene upregulation of IL-in a time-dependent manner similar to that of TGFβ stimulated STAT3 tyrosine phosphorylation. Inhibition of IL-6 with the mIL-6 Receptor Fusion Protein Inhibitor (mIL-6.RFPI) abrogated TGFβ stimulation of STAT3 tyrosine phosphorylation. Further, TGFβ treatment is sufficient to induce cellular invasion in NMuMG cells. This effect was recapitulated by IL-6 treatment, while IL-6 inhibition markedly reduced TGFβ stimulated invasion. The human breast cancer cell lines MDA-MB-231 and MDA-MB-361, which are basal-like and luminal breast cancer models, respectively, display varying levels of constitutive STAT3 tyrosine phosphorylation. The MDA-MB-361 cell line exhibits no endogenous STAT3 phosphorylation but responds to TGFβ treatment in the same manner as nontransformed NMuMG mammary epithelial cells. Conversely, the MDA-MB-231 cell line displays high levels of constitutive STAT3 tyrosine phosphorylation, which data suggest is dependent in part on TGFβ signaling and is completely dependent on IL-6 signaling. Taken together, interruption of the TGFβ-IL-6-STAT3 signaling loop in MDA-MB-231 cells is sufficient to abrogate this signaling and dramatically reduce endogenous cellular invasiveness, while induction of the TGFβ-IL-6-STAT3 pathway in MDA-MB-361 cells confers an invasive phenotype on this otherwise noninvasive cell line. Citation Information: Cancer Res 2009;69(23 Suppl):A17.

GATA-3 Links Tumor Differentiation and Dissemination in a Luminal Breast Cancer Model

How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.