Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells

Abstract

Carcinoma cells residing in an intermediate phenotypic state along the epithelial-mesenchymal (E-M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104⁺/CD44^hi antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably-both in vitro and in vivo-in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E-M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E-M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.

Keywords: E/M hybrid state; EMT; EMT-TFs; Wnt signaling; cancer stem cells.

Fig. 1.

The stable hybrid E/M state is enriched for CSCs and is maintained in vivo. (A) FACS profiles for CD104 and CD44 of HMLER E, E/M, and xM populations. (B) Representative images of E, E/M, and xM cells by phase contrast (brightfield) and IF staining for Zeb1 and E-Cad/Vim/Krt8/18. Nuclei are visualized by DAPI. (C) Representative Western blot analysis of E, E/M, and xM cells for epithelial and mesenchymal markers of three independent lysates. (D) Quantitative PCR assessing levels of mRNA encoding various markers of the E, E/M, and M phenotypic states. (E) Model of the different E–M cell states based on the findings in Results. Snail and Zeb1 indicate the EMT-TFs that are expressed at highest levels (in the E/M state in the case of Snail) or nuclear localization (in the xM state in the case of Zeb1). (F) Assessment by tumor weight of tumorigenicity and tumor growth ability of 1 × 10⁵ E, E/M, and xM cells injected orthotopically. Xenografts were extracted and analyzed 8 wk postimplantation. (G) Analysis of E, E/M, and xM tumor sections using H&E and IF staining for Krt/Zeb1. SV40 LgT staining was used to differentiate tumor cells from mouse stromal cells. Nuclei are visualized by DAPI staining. (H) FACS analysis for CD104 and CD44 expression of dissociated neoplastic cells of E, E/M, and xM tumors. (I) Quantitative analysis of tumor cells that have altered their original in vitro CD104/CD44 marker profile while growing in vivo over a period of 8 wk. ****P < 0.00005 (two-tailed t test). Data are presented as mean ± SEM. Scale bars, brightfield; H&E, 10 µm; IF, 1 µm in B and 2 µm in G.

Fig. 2.

Blocking plasticity reduces tumor formation and growth in xE and xM cells; this inability cannot be compensated by mixing xE and xM cells before implantation into an orthotopic site. (A) Representative Western blot analysis of parental HMLER and trapped xE/xM populations, E, E-SCC-Zeb1KO (EZ^KO), M, and xM-SCCZeb1 (MZ) for epithelial and mesenchymal markers (SCC, single-cell clone). (B) FACS profiles for CD104 and CD44 of E, E-SCC-Zeb1KO, M, and xM-SCCZeb1 populations before implantation into host mice. (C) Assessment of tumorigenicity and tumor growth by tumor weight of orthotopic injection of E, EZ^KO, M, MZ, and 1:1 mix of the trapped xE and xM populations. Data are presented as mean ± SEM. (D) Analysis of E, E-SCC-Zeb1KO, M, and xM-SCCZeb1 and 1:1 mix of the trapped xE and xM tumor sections using H&E. (E) FACS profiles for CD104 and CD44 of E, E-SCC-Zeb1KO, M, and xM-SCCZeb1 expression of dissociated neoplastic cells after they had grown in vivo for 8 wk. (Scale bars, 10 µm.)

Fig. 3.

Zeb1 is needed for a complete EMT, but the hybrid E/M cell state is sufficient for tumor formation. (A) Representative images of cell morphology of E-SCC, E-SCCSn, E-SCC-Zeb1KO, and E-SCC-Zeb1KOSn cells by phase contrast (brightfield) microscopy. (B) FACS profiles for CD104 and CD44 of E-SCC, E-SCCSn, E-SCC-Zeb1KO, and E-SCC-Zeb1KOSn populations. (C) Assessment of tumorigenicity and tumor growth by tumor weight of orthotopic injection E-SCC, E-SCCSn, E-SCC-Zeb1KO, and E-SCC-Zeb1KOSn populations. Data are presented as mean ± SEM. (D) Differences in tumor-initiating ability of E-SCC, E-SCCSn, E-SCC-Zeb1KO, and E-SCC-Zeb1KOSn cells upon transplantation at limiting dilutions into NOD/SCID mice. (E) Analysis of E-SCC, E-SCCSn, E-SCC-Zeb1KO, and E-SCC-Zeb1KOSn tumor sections using H&E and IF staining for Krt/Zeb1 or Snail. LgT staining was used to differentiate tumor cells from mouse stromal cells. Nucleus is visualized by DAPI staining. (Scale bars, brightfield; H&E, 10 µm; IF, 2 µm in E.)

Fig. 4.

Canonical Wnt signaling is active in the E/M state, whereas the xM state is maintained through Wnt5a-driven noncanonical Wnt/PCP signaling. (A) Western blot analysis of E, E/M, and xM cells for canonical Wnt7A/B and noncanonical Wnt5A ligand expression. (B) Assessment of tumor growth by tumor weight after treatment without and with Dox (D) used to induce expression of Fzd8-CRD induction in implanted epithelial (E) cells for 4 wk. Data are presented as mean ± SEM. (C) Analysis of tumor sections using H&E of E tumors ± Dox treatment for Fzd8-CRD induction. (D) Western blot analysis of cell fractionations for E, E/M, and xM cells for canonical Wnt pathway regulators and activated downstream targets. Lamin A/C staining is used as nuclear and CoxIV as cytoplasmic loading control. (E) Western blot analysis of E, E/M, and xM cells for canonical Wnt pathway-activated downstream targets, using CoxIV and GAPDH as loading control. (F) Western blot analysis PAK-PBD agarose bead pull-down assay to visualize activated Rac1 for E, E/M, and xM cells. Lysate of the positive control sample (C) was pretreated with GTPγS. (G) Western blot analysis of E, E/M, and xM cells for noncanonical Wnt pathway-activated downstream targets. (H) Western blot analysis of Wnt5A KD in xM cells. (I) FACS profiles for CD104 and CD44 of xM shLacZ, xM sh1Wnt5A, and xM sh2Wnt5A cell populations. (Scale bars, H&E, 10 µm.)