Project description:In order to metastasize, few cells need to detach from the primary tumor, and transition through the circulation to distant sites to establish new solid tumor metastases. The gene expression changes occurring between these transitions are poorly understood, and controversial findings have been reported regarding transitions between epithelial (E) and mesenchymal (M) cell states. To model the metastasis process in vitro, here we examined the dynamics of gene expression changes and respective cell fates that are occurring in cells that are transitioning from adhesion culture to suspension 3D culture (mammospheres) and back to adhesion culture in vitro. We found that mammosphere and replating cultures of epithelial (E) cell lines (HP cells and E clones) led to enrichment for mesenchymal (M) signatures characteristic of the mesenchymal cell lines, indicative of an epithelial to mesenchymal transition (EMT), but retainment of E-specific signatures (Grosse-Wilde et al., 2015). This co-expression of E and M signatures was stably maintained between the mammosphere state in suspension and in replated cells, indicating a stable E/M gene expression state by partial EMT in E cell lines, which has been associated with a stem-like state (Grosse-Wilde et al., 2015; Jolly et al., 2014; Lu et al., 2014; Zhang et al., 2014). Interestingly, mammosphere suspension culture of mesenchymal M clones also resulted in co-expression of E and M signatures generated by partial mesenchymal to epithelial transition (MET). However, in stark contrast to E clones and HP cells, immediately upon replating M cell-derived mammospheres to adhesion lost the intermediate E/M state and converted back to stable expression of M signatures indicative of rapid EMT. This suggests that the intermediate state in M cells is very unstable. Together, these replating data suggest that prolonged detachment of epithelial-derived cells leads to loss of expression of E signatures and EMT. Life-threatening macrometastases typically recapitulate the gene expression of the primary tumor, which requires expression of E signatures, while the mesenchymal micrometastases are frequent but not-proliferative and harmless. Since all HMLER cell lines independent of their initial E or M state converged to an M state upon expansion in suspension, our data suggest that induction of EMT in suspended HMLER cells may be one reason for the observed benign non-metastatic character of HMLER cells when used in mouse xenograft experiments, and for their metastatic inefficiency. Our datasets of the replating kinetic may be useful to identify which pathways are involved in the processes enabling the mixed HP population to maintain a stable E/M expression signature and a more stem-like state, which we have identified as caused by cell-cell cooperation between phenotypically different E and M cell-types. By contrast, M cells can only transiently assume this E/M gene expression state, and not generate a stable phenotypic heterogeneity and therefore absence of cooperation between E and M cell-types. Instability of the intermediate stem-like E/M state in M cell-types and rapid conversion to the M state can explain why 1) pure M populations are often phenotypically stable and are not undergoing MET in vitro (Schmidt et al., 2015; Zhang et al., 2014), 2) why pure M populations are less aggressive resulting in less metastases than mixed cooperating E and M cell populations (Ocaña et al., 2012; Tsuji et al., 2008) or 3) why complete EMT results in more benign less metastasizing tumors than incomplete EMT (Tran et al., 2014; Tsai et al., 2012) and finally 4) why expression of M traits in primary tumors are associated with good patient outcomes and less metastases than E signatures (Kim et al., 2011; Mylona et al., 2008; Tan et al., 2014). Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J., and Wicha, M.S. (2003). In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17, 1253–1270. Elenbaas, B., Spirio, L., Koerner, F., Fleming, M.D., Zimonjic, D.B., Donaher, J.L., Popescu, N.C., Hahn, W.C., and Weinberg, R.A. (2001). Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 15, 50–65. Grosse-Wilde, A., Fouquier d’Hérouël, A., McIntosh, E., Ertaylan, G., Skupin, A., Kuestner, R.E., del Sol, A., Walters, K.-A., and Huang, S. (2015). Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival. PLOS ONE 10, e0126522. Jolly, M.K., Huang, B., Lu, M., Mani, S.A., Levine, H., and Ben-Jacob, E. (2014). Towards elucidating the connection between epithelial-mesenchymal transitions and stemness. J. R. Soc. Interface R. Soc. 11, 20140962. Kim, H.J., Kim, M.-J., Ahn, S.H., Son, B.H., Kim, S.B., Ahn, J.H., Noh, W.C., and Gong, G. (2011). Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status. The Breast 20, 78–85. Lu, M., Jolly, M.K., Onuchic, J., and Ben-Jacob, E. (2014). Toward decoding the principles of cancer metastasis circuits. Cancer Res. 74, 4574–4587. Mylona, E., Giannopoulou, I., Fasomytakis, E., Nomikos, A., Magkou, C., Bakarakos, P., and Nakopoulou, L. (2008). The clinicopathologic and prognostic significance of CD44+/CD24−/low and CD44−/CD24+ tumor cells in invasive breast carcinomas. Hum. Pathol. 39, 1096–1102. Ocaña, O.H., Córcoles, R., Fabra, Á., Moreno-Bueno, G., Acloque, H., Vega, S., Barrallo-Gimeno, A., Cano, A., and Nieto, M.A. (2012). Metastatic Colonization Requires the Repression of the Epithelial-Mesenchymal Transition Inducer Prrx1. Cancer Cell 22, 709–724. Schmidt, J.M., Panzilius, E., Bartsch, H.S., Irmler, M., Beckers, J., Kari, V., Linnemann, J.R., Dragoi, D., Hirschi, B., Kloos, U.J., et al. (2015). Stem-Cell-like Properties and Epithelial Plasticity Arise as Stable Traits after Transient Twist1 Activation. Cell Rep. 10, 131–139. Tan, T.Z., Miow, Q.H., Miki, Y., Noda, T., Mori, S., Huang, R.Y.-J., and Thiery, J.P. (2014). Epithelial-mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients. EMBO Mol. Med. 6, 1279–1293. Tran, H.D., Luitel, K., Kim, M., Zhang, K., Longmore, G.D., and Tran, D.D. (2014). Transient SNAIL1 Expression Is Necessary for Metastatic Competence in Breast Cancer. Cancer Res. 74, 6330–6340. Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal Regulation of Epithelial-Mesenchymal Transition Is Essential for Squamous Cell Carcinoma Metastasis. Cancer Cell 22, 725–736. Tsuji, T., Ibaragi, S., Shima, K., Hu, M.G., Katsurano, M., Sasaki, A., and Hu, G. -f. (2008). Epithelial-Mesenchymal Transition Induced by Growth Suppressor p12CDK2-AP1 Promotes Tumor Cell Local Invasion but Suppresses Distant Colony Growth. Cancer Res. 68, 10377–10386. Zhang, J., Tian, X.-J., Zhang, H., Teng, Y., Li, R., Bai, F., Elankumaran, S., and Xing, J. (2014). TGF- -induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci. Signal. 7, ra91–ra91. For these analyses we used the heterogeneous parental HMLER (HP) cell line, with isogenic epithelial (E) and mesenchymal (M) cell-types (Elenbaas et al., 2001), and stable clonal HMLER-derived E and M cell lines, which are normally passaged under adhesion conditions (_adh, GSE66527). We cultured cell lines under 3D suspension conditions resembling mammosphere growth conditions that are typically used to enrich for stem-like cells (Dontu et al., 2003), and examined gene expression of cells under mammosphere conditions (_sus, GSE66527), or after replating (_re) in a kinetic examining gene expression from 6, 24, 96, and 240 hrs after cells were allowed to readhere to normal dishes for the heterogeneous but mostly epithelial (HP) cell lines and mesenchymal (M4) cell lines. To validate our findings we also examined gene expression of cells replated after mammosphere culture for about two weeks from three different E (E3, E4, E5) and M clones (M3, M4, M1). All gene expression samples were taken from biological replicates (at least duplicates) of cells grown in different wells or even at different times. Gene expression of replated (_re) cells was compared to the respective cell lines grown under adhesion conditions (_adh), as well as to the respective cell line grown under suspension mammosphere condition (_sus) which have been deposited previously under accession number GSE66527. HP replicates (re): HP_re_AGW120913, HP_re_RK12026, HP_re_AGW1209_5, HP_re_AGW1209_6, HP_re_AGW1209_7, HP_re_AGW1209_8, HP_re_AGW1209_9, HP_re_0607_1, HP_re_0607_2 E3 replicates (re): E3_re_RK1202, E3_re_RK12028; E4 replicates: E4_re_0622, E4_re_0613; E5 replicates: E5_re_0613_1, E5_re_0613_2 M1 replicates (re): M1_re_0607_1, M1_re_0607_2, M2 replicates: M2_re_RK1202, M2_re_RK12029, M2_re_AGW1209_3, M2_re_AGW1209_4, M2_re_AGW1209_5, M3 replicates: M4_re_0607_1, M4_re_0607_2 HP replicates (adh): HP_adh_0629, HP_adh_2210, HP_adh_0607sc, HP_adh_0607c E3 replicates (adh): E3_adh_0629, E3_adh, E3_adh_0826. E4 replicates (adh): E4_adh_0607, E4_adh_0210, E5 replicates (adh): E5_adh_0210, E5_adh_0607 M1 replicates (adh): M1_adh_0210, M1_adh_0607, M4 replicates (adh): M4_adh_0607, M4_adh_0210, M3 replicates (adh): M3_adh, M3_adh_2210, M3_adh_2810 HP replating kinetic (adh): HP_adh_0607c (GSE66527), HP_adh_0607sc (GSE66527), HP (mammospheres, suspension): HP_sus_0603_2 (GSE66527), HP_sus_0603_1 (GSE66527), HP (replated 6 hrs): HP_re_6_0603_1, HP_re_6_0603_2, HP (replated 24hrs): HP_re_24_0607_1, HP_re_24_0607_2, HP (replated 96 hrs): HP_re_96_0607_1, HP_re_96_0607_2, HP (replated 240hrs): HP_re_0607_1, HP_re_0607_2, M4 replating kinetic (adh): M4_adh_0210 (GSE66527), M4_adh_0607 (GSE66527), M4 (mammospheres, suspension): M4_sus_0603_2 (GSE66527), M4_sus_0603_1 (GSE66527), M4 (replated 24 hrs): M4_re_24_0603_1, M4_re_24_0603_2, M4 (replated 96 hrs), M4_re_96_0607_1, M4_re_96_0607_2, M4 replated 240hrs): M4_re_0607_1, M4_re_0607_2

Project description:Background Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. Methods To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. Results Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. Conclusions We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Project description:TSV40ER and hTERT immortalized mammospheres undergo transformation in culture to generate breast cancer stem cells The stem cell hypothesis of cancer predicts that cancers arise from the transformation of normal stem/progenitor cells present within adult tissues. However, the molecular mechanisms that lead to the transformation of these cells remain largely unknown, mainly owing to the lack of suitable model systems. In this study, we have generated an immortalized breast epithelial cell line, NBLE, by over-expressing the SV40 Early Region and the catalytic domain of human telomerase into mammosphere-derived cells that are enriched in breast stem/progenitor cells. While primary mammospheres fail to grow beyond four passages in suspension culture, NBLE cells propagated continuously generating spheres at a high frequency of 35%. These cells also expressed stemness-related genes, and differentiated into both luminal and myoepithelial lineages. Interestingly, after around 135 population doublings in culture, the late passage cells exhibited a mesenchymal morphology and contained >90% of CD44+/CD24Low/- cells resembling breast cancer stem cells. When injected subcutaneously into nude mice, the late passage NBLEs formed invasive tumors closely resembling breast adenocarcinomas, the most common type of breast cancer. The transformed NBLE cells showed hyperactivation of Wnt, Hh and TGFbeta pathways, suggesting that the deregulation of these self-renewal pathways may have in part contributed to tumorigenesis. Gene expression analysis further revealed a high degree of similarity with breast cancer stem cells. Thus, our study provides a unique model system to study signaling pathways involved in self-renewal, differentiation, and transformation of breast stem/progenitor cells. Additionally, the NBLE cells can be utilized for screening drugs specifically targeting breast cancer stem cells. NBLE cells were generated by over-expressing SV40ER and hTERT into mammosphere-derived cells. These cells harnored CD44+/CD24- fraction that resembled breast cancer stem cells. To understand the unique gene expression of this fraction we performed microarray on CD44+/CD24- versus rest fractions from NBLE cells (Rep 1 and Rep 2). To see if this gene profile has any similarity with breast cancer stem cells, we performed CD44+/CD24- versus rest microarray on two primary breast tumors (CB272 and CB258-Rep1 andRep2).

Project description:In order to metastasize, few cells need to detach from the primary tumor, and transition through the circulation to distant sites to establish new solid tumor metastases. The gene expression changes occurring between these transitions are poorly understood, and controversial findings have been reported regarding transitions between epithelial (E) and mesenchymal (M) cell states. To model the metastasis process in vitro, here we examined the dynamics of gene expression changes and respective cell fates that are occurring in cells that are transitioning from adhesion culture to suspension 3D culture (mammospheres) and back to adhesion culture in vitro. We found that mammosphere and replating cultures of epithelial (E) cell lines (HP cells and E clones) led to enrichment for mesenchymal (M) signatures characteristic of the mesenchymal cell lines, indicative of an epithelial to mesenchymal transition (EMT), but retainment of E-specific signatures (Grosse-Wilde et al., 2015). This co-expression of E and M signatures was stably maintained between the mammosphere state in suspension and in replated cells, indicating a stable E/M gene expression state by partial EMT in E cell lines, which has been associated with a stem-like state (Grosse-Wilde et al., 2015; Jolly et al., 2014; Lu et al., 2014; Zhang et al., 2014). Interestingly, mammosphere suspension culture of mesenchymal M clones also resulted in co-expression of E and M signatures generated by partial mesenchymal to epithelial transition (MET). However, in stark contrast to E clones and HP cells, immediately upon replating M cell-derived mammospheres to adhesion lost the intermediate E/M state and converted back to stable expression of M signatures indicative of rapid EMT. This suggests that the intermediate state in M cells is very unstable. Together, these replating data suggest that prolonged detachment of epithelial-derived cells leads to loss of expression of E signatures and EMT. Life-threatening macrometastases typically recapitulate the gene expression of the primary tumor, which requires expression of E signatures, while the mesenchymal micrometastases are frequent but not-proliferative and harmless. Since all HMLER cell lines independent of their initial E or M state converged to an M state upon expansion in suspension, our data suggest that induction of EMT in suspended HMLER cells may be one reason for the observed benign non-metastatic character of HMLER cells when used in mouse xenograft experiments, and for their metastatic inefficiency. Our datasets of the replating kinetic may be useful to identify which pathways are involved in the processes enabling the mixed HP population to maintain a stable E/M expression signature and a more stem-like state, which we have identified as caused by cell-cell cooperation between phenotypically different E and M cell-types. By contrast, M cells can only transiently assume this E/M gene expression state, and not generate a stable phenotypic heterogeneity and therefore absence of cooperation between E and M cell-types. Instability of the intermediate stem-like E/M state in M cell-types and rapid conversion to the M state can explain why 1) pure M populations are often phenotypically stable and are not undergoing MET in vitro (Schmidt et al., 2015; Zhang et al., 2014), 2) why pure M populations are less aggressive resulting in less metastases than mixed cooperating E and M cell populations (Ocaña et al., 2012; Tsuji et al., 2008) or 3) why complete EMT results in more benign less metastasizing tumors than incomplete EMT (Tran et al., 2014; Tsai et al., 2012) and finally 4) why expression of M traits in primary tumors are associated with good patient outcomes and less metastases than E signatures (Kim et al., 2011; Mylona et al., 2008; Tan et al., 2014). Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J., and Wicha, M.S. (2003). In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17, 1253–1270. Elenbaas, B., Spirio, L., Koerner, F., Fleming, M.D., Zimonjic, D.B., Donaher, J.L., Popescu, N.C., Hahn, W.C., and Weinberg, R.A. (2001). Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 15, 50–65. Grosse-Wilde, A., Fouquier d’Hérouël, A., McIntosh, E., Ertaylan, G., Skupin, A., Kuestner, R.E., del Sol, A., Walters, K.-A., and Huang, S. (2015). Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival. PLOS ONE 10, e0126522. Jolly, M.K., Huang, B., Lu, M., Mani, S.A., Levine, H., and Ben-Jacob, E. (2014). Towards elucidating the connection between epithelial-mesenchymal transitions and stemness. J. R. Soc. Interface R. Soc. 11, 20140962. Kim, H.J., Kim, M.-J., Ahn, S.H., Son, B.H., Kim, S.B., Ahn, J.H., Noh, W.C., and Gong, G. (2011). Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status. The Breast 20, 78–85. Lu, M., Jolly, M.K., Onuchic, J., and Ben-Jacob, E. (2014). Toward decoding the principles of cancer metastasis circuits. Cancer Res. 74, 4574–4587. Mylona, E., Giannopoulou, I., Fasomytakis, E., Nomikos, A., Magkou, C., Bakarakos, P., and Nakopoulou, L. (2008). The clinicopathologic and prognostic significance of CD44+/CD24−/low and CD44−/CD24+ tumor cells in invasive breast carcinomas. Hum. Pathol. 39, 1096–1102. Ocaña, O.H., Córcoles, R., Fabra, Á., Moreno-Bueno, G., Acloque, H., Vega, S., Barrallo-Gimeno, A., Cano, A., and Nieto, M.A. (2012). Metastatic Colonization Requires the Repression of the Epithelial-Mesenchymal Transition Inducer Prrx1. Cancer Cell 22, 709–724. Schmidt, J.M., Panzilius, E., Bartsch, H.S., Irmler, M., Beckers, J., Kari, V., Linnemann, J.R., Dragoi, D., Hirschi, B., Kloos, U.J., et al. (2015). Stem-Cell-like Properties and Epithelial Plasticity Arise as Stable Traits after Transient Twist1 Activation. Cell Rep. 10, 131–139. Tan, T.Z., Miow, Q.H., Miki, Y., Noda, T., Mori, S., Huang, R.Y.-J., and Thiery, J.P. (2014). Epithelial-mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients. EMBO Mol. Med. 6, 1279–1293. Tran, H.D., Luitel, K., Kim, M., Zhang, K., Longmore, G.D., and Tran, D.D. (2014). Transient SNAIL1 Expression Is Necessary for Metastatic Competence in Breast Cancer. Cancer Res. 74, 6330–6340. Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal Regulation of Epithelial-Mesenchymal Transition Is Essential for Squamous Cell Carcinoma Metastasis. Cancer Cell 22, 725–736. Tsuji, T., Ibaragi, S., Shima, K., Hu, M.G., Katsurano, M., Sasaki, A., and Hu, G. -f. (2008). Epithelial-Mesenchymal Transition Induced by Growth Suppressor p12CDK2-AP1 Promotes Tumor Cell Local Invasion but Suppresses Distant Colony Growth. Cancer Res. 68, 10377–10386. Zhang, J., Tian, X.-J., Zhang, H., Teng, Y., Li, R., Bai, F., Elankumaran, S., and Xing, J. (2014). TGF- -induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci. Signal. 7, ra91–ra91.

Dataset Information

Identification of signatures specific to the epithelial or mesenchymal phenotypes from the heterogeneous mammary epithelial HMLER cells

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