Beyond retinoblastoma, RB1
is also frequently mutated in osteosarcoma and small-cell lung cancer, and patients heterozygous for RB1
have significantly increased risk for developing these cancers (Sábado Alvarez, 2008
; Kansara and Thomas, 2007
). Indeed, tumors derived from TKO and RB1−/−
MEFs contained cells resembling spindle cell sarcomas. Moreover, the RB1 family is regulated by phosphorylation mediated by cdks, and in tumors where RB1
is not mutated the pathway is inactivated by hyperphosphorylation of family members (Kaye, 2002
; Du and Pogoriler, 2006
; Frolov and Dyson, 2004
; Lowe and Sherr, 2003
). Thus, loss of the RB1 pathway appears to be a hallmark of most if not all cancers. The loss of cell-cycle control when the RB1 pathway is disrupted is thought to play an important role in the initiation of tumor cell proliferation. However, beyond tumor expansion, we propose that the loss of cell contact inhibition when the RB1 pathway is inhibited leads to outgrowth into sphere-like structures where cell-cell contacts predominate, and these conditions in the advancing cancer trigger reprogramming of somatic cells to cells with properties of cancer stem cells (). Even though RB1−/−
MEFs were contact inhibited in monolayer culture, they could still form spheres when placed in suspension culture and generate SP cells with properties of cancer stem cells. While similar spheres formed with wild-type MEFs and differentiated cells were generated in these spheres (results not shown), neither these wild-type spheres nor cells derived from the spheres were able to form tumors in nude mice, emphasizing the importance of RB1−/−
mutation in generation of tumor cells in the spheres. Our studies are consistent with previously published results implying that cells with properties of cancer stem cells can be generated from somatic cells, and they suggest a pathway linked to tumor outgrowth for generation of such cells in more advanced tumors.
Interestingly, the SP cells with properties of cancer stem cells isolated from spheres of MEFs mutant in the RB1 pathway expressed ESC genes including Oct-4, Sox-2, Nanog, Klf4, and c-myc
, and this was accompanied by induction of genes in pathways known to be important for ESCs maintenance such as TFG-β/BMP, Wnt, Notch, hedgehog, and FGF. To our knowledge, this is the first example that silenced endogenous ESC genes such as Oct-4 and Nanog can be reactivated in differentiated somatic cells. Beyond production of cancer cells, a number of cells in TKO and RB1−/−
MEF spheres express markers representative of all three embryonic layers, reminiscent of what occurs in embryoid bodies derived from ESCs. It has been demonstrated previously that cancer stem cells can also give rise to differentiated cells along with cancer cells (Dirks, 2008
). While we are unsure of the steps involved in the differentiation process in the spheres, it is interesting to speculate that sphere formation is reprogramming differentiated somatic cells with a mutant RB1 pathway back to a multipotential phenotype capable of generating differentiated cells, and that these progenitor cells correspond to the SP. In support of this notion, isolated SP cells could reform spheres and again induce ESC genes and a series of differentiation markers. Furthermore, injection of SP cells into nude mice led not only to production of what appeared to be a homologous population of cancer cells (spindle cell sarcoma), but also to neuronal cells segregated in whorls in a niche distinct from the cancer cells. While wild-type ESCs form teratomas when injected into nude mice, it is of note that TKO ESCs formed spherical structures expressing early neuronal markers in nude mice (Dannenberg et al., 2000
). In this way, the SP cells derived from TKO and RB1−/−
MEF spheres appear to resemble TKO ESCs.
Recent studies have demonstrated that overexpression of EMT transcription factors in epithelial cells can trigger the CD44-high/CD24-low expression pattern that is thought to be important for generation of breast cancer stem cells (Mani et al., 2008
). We also observed induction of CD44 and diminished expression of CD24 mRNAs in SP cells. While we did not see a change in expression of the EMT factors Snai1, Snai2, or Zeb2, Zeb1 mRNA expression was induced in SP versus MP cells. Additionally, this elevated expression of Zeb1 was accompanied by repression of E-cadherin and induction of smooth muscle actin in the SP cells, suggesting a relatively mesenchymal expression pattern compared to MP cells. Zeb1 is directly repressed by E2F-RB1 family complexes, which appears to limit its expression to proliferating cells in vivo (Liu et al., 2007
), and the downregulation of RBL1 and RBL2 initially during sphere formation may facilitate this induction during sphere formation.
Inspection of the CD24 promoter region revealed multiple consensus Zeb1 binding sites, suggesting that CD24 may be a direct target of Zeb1 repression in SP cells. Indeed, knockdown of Zeb1 but not Zeb2 led to induction of CD24 mRNA, suggesting that Zeb1 is important in repressing CD24 expression. Zeb1 expression is tightly linked to cell proliferation in vivo, and it directly represses the cdk inhibitors p15INK4b and p21CDKN1a, which inhibit the cell cycle (Liu et al., 2008
and references therein). Heterozygous mutation of Zeb1
is sufficient for induction of both of these genes, and it leads to premature senescence of MEFs in culture, and induction of p15INK4b in Zeb1−/−
cells in vivo is linked to diminished mesenchymal and CNS progenitor cell proliferation during development with accompanying developmental defects (Liu et al., 2008
). Knockdown of Zeb1 inhibited growth of spheres in suspension, it prevented generation of MP cells from isolated SP cells, and it led to the eventual loss of SP cell viability in culture. In this regard, it is of note that the Drosophila
homolog of Zeb1, Zfh1, was recently shown to be necessary for viability of stem cells in the testis (Leatherman and Dinardo, 2008
), implying an important role in normal stem cell viability.