In this study we demonstrate that mesenchymal-like malignant subpopulations contribute to chemotherapy resistance in SCCs with predominantly epithelial differentiation. Previous studies correlated drug resistance in carcinoma lines with increased expression of mesenchymal markers (Frederick et al., 2007
; Ghoul et al., 2009
; Shrader et al., 2007
; Thomson et al., 2005
; Yang et al., 2006
; Yauch et al., 2005
). However, analyzing lines as total populations does not account for the possible presence of epithelial and mesenchymal-like subsets, which may make disparate contributions to overall drug resistance. We suggest that SCCs in vivo
typically do not possess uniformly epithelial or mesenchymal-like phenotypes; accordingly, the heterogeneity defined here in SCC cell lines was maintained by their in vivo
counterparts. Consistent with this observation, a study examining several HNSCC histologic types revealed heterogeneous vimentin staining to be typical, with only a rare subtype, the “sarcomatoid” carcinoma, expressing uniformly high levels throughout (Mandal et al., 2008
). At the other end of the spectrum, cell lines with high E-cadherin expression can still contain small vimentin-expressing subsets (Figure S1
), and we note the same observation in xenografts of human HNSCC specimens (unpublished data). Nevertheless, it is unlikely that the precise markers used here to define the treatment resistant subpopulation will identify subsets with similar properties across all SCCs. We speculate that more subtle phenotypic transitions sharing key molecular traits with the ones documented here may be a basis for intrinsic treatment resistance across many SCC tumors.
In this study, intrinsic paclitaxel resistance of the mesenchymal-like subpopulation seemed attributable to its slower growth. However, cytotoxic drug resistance after EMT is likely more than a direct function of growth rate and instead reflects the outcome of gene-regulatory programs with complex relationships to proliferation, differentiation, and apoptosis. For instance, a recent study showed that paclitaxel induces snail and slug, which, in addition to being transcription factor mediators of EMT, also repressed promoters of key molecules involved in p53-mediated apoptosis (Kurrey et al., 2009
). Thus, at least in vitro,
paclitaxel likely depletes the epithelial subset by multiple mechanisms and may also contribute to enrichment of mesenchymal-like cells by promoting EMT. Similarly, adriamycin, a cytotoxic drug of differing mechanism, also enhances EMT and creates resistance to itself and other agents as a twist1-mediated phenomenon (Li et al., 2009b
Resistance to cetuximab in the Ecad-lo subset here likely arose from reduced EGFR expression and resulting loss of MAPK and PI3K pathway regulation by the receptor. This correlation contrasts with the lack of positive association between EGFR over-expression and clinical cetuximab sensitivity in HNSCC (Kalyankrishna and Grandis, 2006
). Our findings also differ with a study of hepatocellular carcinoma cell lines, where EGFR levels appeared independent of mesenchymal marker expression (Fuchs et al., 2008
). However, EGFR expression had thus far been compared against mesenchymal features using bulk lines, where overall receptor levels may not reflect those of any mesenchymal-like subsets they contain.
Known resistance mechanisms to EGFR-targeted therapies (Bianco et al., 2005
; Cooper and Cohen, 2009
) provide context for understanding the cetuximab insensitivity observed here. A novel mechanism for acquired cetuximab resistance is ligand-independent EGFR function through down-regulation of surface receptor and elevated nuclear, cytoplasmic, and total receptor levels (Li et al., 2009a
). This mechanism may partly explain the association in HNSCCs of EGFR gene amplification and poor prognosis (Chung et al., 2006
; Temam et al., 2007
), but the reduced surface and total EGFR here in Ecad-lo cells argues against this explanation for their resistance. An alternative explanation is switching to other receptor tyrosine kinases to restore downstream survival and proliferation signals. For instance, EGFR and ErbB2 expression were noted to segregate to different regions within ESCCs (Kawaguchi et al., 2007
) and thus might serve comparable functions in distinct subpopulations. Also, EGFR down-regulation and coordinate up-regulation of ErbB2/ErbB3 in HNSCC are described in response to the EGFR-targeted small molecule gefitinib (Erjala et al., 2006
). Kinase switching to receptors outside the EGFR family is observed in mesenchymal-like cell lines of non small cell lung cancers (NSLC) (Thomson et al., 2005
) and is a plausible mechanism here. Notably, the EGFR activating mutations that sensitize NSLC to gefitinib (Gazdar, 2009
) are largely absent in HNSCC (Temam et al., 2007
), and thus sensitivity to this inhibitor is not anticipated in the face of cetuximab resistance.
Traits of the mesenchymal-like subpopulation in SCCs have implications in the wider discussion regarding origins of phenotypic heterogeneity within solid tumors. Specifically, it remains unclear how much intra-tumor heterogeneity arises from epigenetic differences versus genetic change driven by clonal evolution (Shackleton et al., 2009
). The ability of epithelial and mesenchymal-like subsets to reconstitute each other from single cell clones, as shown here, suggests that these phenotypes are distinguished primarily by epigenetic alterations and not genotypic selection. The cancer stem cell hypothesis is one framework for explaining how an epigenetically distinct subpopulation may sustain tumor growth and resist drug therapy. We have elected here not to frame the mesenchymal-like subset explicitly within controversial and evolving definitions of cancer stem cells; however, this subset appears to maintain malignant potential and does not represent a terminally differentiated phenotype. The subpopulation in SCC may have functions analogous to the CD44+/CD24− stem-like subset in breast carcinoma on the basis of sharing its mesenchymal-like gene expression profile (Mani et al., 2008
). If so, the in vitro
plasticity of our subpopulations argues against a rigid developmental hierarchy originating with stem cells and ending in terminal differentiation, instead favoring one where phenotypes are dynamic and sensitive to microenvironments. It is possible that the mesenchymal-like subset in SCCs retains the phenotypic plasticity to repopulate other subpopulations post therapy.
In context of our findings, promoting phenotypic reversal of Ecad-lo cells through mesenchymal to epithelial transition (MET) may be a viable strategy for chemosensitization. Here pure mesenchymal-like clones of SCC lines showed initial resistance to MET but did restore the epithelial subpopulation spontaneously after extended culture. Experimental maneuvers that both promote MET and enhance drug sensitivity include down-regulation of Notch (Wang et al., 2009
), induction of Raf-kinase inhibitor protein (Wu and Bonavida, 2009
), knockdown of zeb-1 (Haddad et al., 2009
), and induction of miR-200 microRNA family members (Adam et al., 2009
; Gregory et al., 2008
; Li et al., 2009c
). Future studies will seek pharmacologic strategies for both specifically targeting the mesenchymal-like subpopulation in SCCs and promoting its phenotypic reversal.