Prior studies correlate the presence of mesenchymal traits in carcinoma cell lines with resistance to both conventional cytotoxic11,12
and EGFR inhibitory13–15
agents. Our previous work has identified similar resistance to both classes of compounds in an Ecad-lo, mesenchymal-like subpopulation present within individual SCC cell lines.6
In this study, we demonstrate that salinomycin has efficacy against the mesenchymal-like subpopulation of SCC cells that is resistant to cisplatin, the single most widely used cytotoxic agent for HNSCC.16
Salinomycin was highly toxic in vitro to both Ecad-hi and Ecad-lo subpopulations but only modestly selective for Ecad-lo vs. Ecad-hi cells. This finding contrasts with salinomycin's more striking depletion of vimentin-positive cells from direct xenograft tumors in vivo. This disparity may be accounted for by the role of complex pharmacodynamics in the in vivo setting in determining treatment outcome. For example, the extended multi-dose regimen used in vivo may have allowed differential repopulation of the dual subsets between doses, thus increasing the size disparity between vimentin-positive and -negative areas in residual tumors post-treatment. The potential for such phenomena is supported by the in vitro data, which shows that surviving Ecad-lo cells after salinomycin treatment retain much less growth potential upon reculture than their cisplatin-treated counterparts.
The efficacy of salinomycin against mesenchymal-like SCC cells in this study adds to prior evidence that it can target treatment- resistant malignant subpopulations within other cancers. In particular, it targets CD44+
cells in breast carcinoma7
as well as c-kitlow
cells with imatinib resistance in a genetic mouse model of gastrointestinal stromal tumors.17
In principle, its efficacy against any given subpopulation may arise not only from direct toxicity to that phenotype, but by altering the dynamic interconversion between epithelial and mesenchymal-like subpopulations. For instance, salinomycin's depletion of Ecad-lo SCC cells may arise through selective killing, inhibition of epithelial-to-mesenchymal transition and/or promotion of mesenchymal to epithelial differentiation. Although salinomycin's toxicity is readily apparent in vitro, it is difficult to confirm or preclude other possible effects on differentiation, particularly in the in vivo DTX model.
In regard to mechanism of action, salinomycin is a potassium-selective ionophore antibiotic whose basis for anti-cancer activity is presently unknown. Its current therapeutic use is limited to live-stock, chiefly as an anticoccidial drug in poultry and to enhance feed efficiency in ruminants.18,19
Salinomycin induces apoptosis by a caspase-independent mechanism across human cancer cell lines of diverse origin.20
This study also showed its diminished effects against normal cells, indicating that salinomycin's high in vitro toxicity here for both Ecad-hi and Ecad-lo subsets is likely a cancer-specific effect. Other reports suggest mechanisms behind salinomycin's apparent capacity to deplete subpopulations resistant to chemotherapy. It appears to reverse resistance mediated by ABC transporter family members21
and may enhance cytotoxic drug efficacy through inhibiting the P-glycoprotein (ABCB1) member of this family.22
The pertinence of such chemosensitizing mechanisms to salinomycin's action in single-drug use is presently unknown.
Our results suggested that the cytotoxic agents and salinomycin may be additive or synergistic in inducing regression of carcinomas containing mixed epithelial and mesenchymal-like phenotypes. Our failure to observe combined efficacy for the two compounds during in vitro testing may be explained by possible overlap or interference between their killing mechanisms. Because salinomycin does not clearly discriminate between Ecad-hi and Ecad-lo subpopulations in vitro, the benefit from adding cisplatin in the absence of complementary mechanisms for Ecad-hi cells may be minimal. In addition, a high in vivo toxicity and narrow therapeutic window for salinomycin were observed in NSG mice, preventing the daily administrations and higher dosing previously described in reference 8
. This narrow therapeutic window hampered efforts to determine usable in vivo doses in combination with cisplatin. Such limitations may be relevant in humans, where major salinomycin toxicity is reported from incidental inhalational exposure.23
Based on these findings, salinomycin seems more likely to remain a prototypic tool compound than to advance into human therapeutic use.
Our detection of vimentin-positive malignant subpopulations across multiple advanced stage human HNSCCs adds new evidence for the physiologic relevance of EMT in carcinoma biology. Whether an EMT-like process commonly occurs in carcinomas in vivo remains controversial,9
with some questioning whether it is primarily an artifact of in vitro cell line differentiation. In this study, the observed maintenance of heterogeneous E-cadherin and vimentin expression within SCC cells after growth as xenografts weighs against this perspective. A barrier to addressing this controversy using clinical carcinoma samples has been the difficulty in distinguishing tumor subpopulations expressing mesenchymal markers from nonmalignant stromal cells of mesenchymal origin.10
After depletion of nonmalignant cells in this study by serial mouse passage of DTXs, xenografts contained human vimentin staining patterns closely recapitulating the staining heterogeneity in their tumors of origin. The maintenance of human vimentin-expressing subpopulations within all six successfully xenografted specimens of advanced metastatic HNSCC increases evidence for a pervasive EMT-like phenomenon in human SCCs. Furthermore, different morphologies seen among vimentin-positive cells in certain xenograft tumors () may potentially represent two distinct modes of migration previously described within cancer cell lines having undergone EMT.24,25
Specifically, a tentative assignment of mesenchymal or amoeboid migration may be made to the respective fusiform and irregular morphologies of vimentin-positive cells seen at the invasive front. More detailed characterization of these subpopulations across a broader panel of tumors remains to be accomplished; however, our present data show that HNSCCs do not divide into the dichotomous epithelial and mesenchymal-like categories implied by previous studies, with cell lines grouped by predominant phenotype.15
Instead, we show a spectrum of mixed epithelial and mesenchymal-like differentiation within individual HNSCCs, as previously suggested to exist within ovarian carcinomas.26
The therapeutic implications of these mixed phenotypes in vivo are highlighted in this study by salinomycin and cisplatin exerting opposite effects on the size of the vimentin-expressing subpopulation in DTXs. The capacity of salinomycin to target mesenchymal-like carcinoma cells also raises the possibility that such compounds could beneficially alter invasive and metastatic behavior, in which EMT likely participates. Overall, these findings underscore a need to develop novel compounds targeted at specific treatment-resistant phenotypes. Their use in combination with existing drugs offers a promising avenue toward achieving curative outcomes using chemotherapy as single-modality treatment.