Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival

Abstract

Breast cancer stem cells (CSCs) are thought to drive recurrence and metastasis. Their identity has been linked to the epithelial to mesenchymal transition (EMT) but remains highly controversial since—depending on the cell-line studied—either epithelial (E) or mesenchymal (M) markers, alone or together have been associated with stemness. Using distinct transcript expression signatures characterizing the three different E, M and hybrid E/M cell-types, our data support a novel model that links a mixed EM signature with stemness in 1) individual cells, 2) luminal and basal cell lines, 3) in vivo xenograft mouse models, and 4) in all breast cancer subtypes. In particular, we found that co-expression of E and M signatures was associated with poorest outcome in luminal and basal breast cancer patients as well as with enrichment for stem-like cells in both E and M breast cell-lines. This link between a mixed EM expression signature and stemness was explained by two findings: first, mixed cultures of E and M cells showed increased cooperation in mammosphere formation (indicative of stemness) compared to the more differentiated E and M cell-types. Second, single-cell qPCR analysis revealed that E and M genes could be co-expressed in the same cell. These hybrid E/M cells were generated by both E or M cells and had a combination of several stem-like traits since they displayed increased plasticity, self-renewal, mammosphere formation, and produced ALDH1+ progenies, while more differentiated M cells showed less plasticity and E cells showed less self-renewal. Thus, the hybrid E/M state reflecting stemness and its promotion by E-M cooperation offers a dual biological rationale for the robust association of the mixed EM signature with poor prognosis, independent of cellular origin. Together, our model explains previous paradoxical findings that breast CSCs appear to be M in luminal cell-lines but E in basal breast cancer cell-lines. Our results suggest that targeting E/M heterogeneity by eliminating hybrid E/M cells and cooperation between E and M cell-types could improve breast cancer patient survival independent of breast cancer-subtype.

Jagged mediates differences in normal and tumor angiogenesis by affecting tip-stalk fate decision

“…The emergence of a hybrid tip/stalk phenotype also lends support to the emerging notion "a black and white distinction between tip and stalk cells is an oversimplification" (1) and strengthens the increasingly accepted notion that a hybrid state that coexpresses markers of two lineages is a signature of enhanced plasticity (multipotency) of a system (41)(42)(43). We find that the tendency to adopt this hybrid phenotype is reduced at high levels of Fringe, a glycosyltransferase that promotes Notch-Delta signaling at the expense of Notch-Jagged signaling by modifying Notch to increase its affinity for Delta and decrease it for Jagged.…”

Survival Outcomes in Cancer Patients Predicted by a Partial EMT Gene Expression Scoring Metric

“…TEM4-18 cells, negative for E-cadherin and displaying nuclear staining for Zeb1 [30], were predicted to be mesenchymal, whereas TEM2-5, with relatively higher levels of cell-adhesion molecules as compared to TEM4-18 [30], were predicted to be hybrid E/M (Table 2A). Similarly, PC-3/Mc cells, a subpopulation of PC-3 cells that co-expressed CD24 and CD44 [31] (a signature of hybrid E/M [9]), were predicted to be hybrid E/M, and PC-3/S cells, being enriched in mesenchymal gene expression [31], were predicted as mesenchymal (Table 2A) (GSE24868). Higher tumor-initiation potential and an active self-renewal program in PC-3/Mc further reinforce the hypothesis that cells in a hybrid E/M state, instead of those frozen in a mesenchymal state, are most likely to be more stem-like [1, 32, 33].…”

“…Of particular interest is the observation that breast cancer patients with lower EMT scores had better overall and progression-free survival, except when investigating a dataset enriched in basal-like breast cancer. These apparent contradictions may result from a combination of factors such as different therapeutic treatments driving phenotypic transitions [39, 50], and methods of generating EMT-specific signature used to classify patients for survival analysis [9]. Prior work has relied on inferring characteristics of the intermediate E/M phenotype by interpolating between known behavior for E and M states [9,38].…”

The impact of melatonin and carbon ion irradiation on cancer stem cells

“…The epithelial mesenchymal transition (EMT) is a process that allows cancer stem cells to become invasive and metastatic [69][70][71]. This process is mediated by the activity of growth and transcription factors, leading to loss of the intercellular junction structure of epithelial cells, obtaining a mesenchymal morphology, loss of apical-basal cell polarity and migration/invasion capability [72][73][74].…”