Oncogene-specific formation of chemoresistant murine hepatic cancer stem cells

Abstract

At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of activation of v-akt murine thymoma viral oncogene homolog (AKT) and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This "side population" (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers, and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ATP binding cassette drug transporter proteins that also contribute to chemoresistance in cancer. We found that the multidrug resistance gene 1 (MDR1) transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1.

Conclusion: The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer.

Fig. 1. MYC-induced hepatic tumors have increased SP cells compared to normal liver

(A) Upper row, gross morphology of livers and tumors from FVB/N Wt, LT2 and LT2-MYC transgenic mice and MYC-transfected mice 8 weeks after removal of doxycycline or hydrodynamic transfection. Lower row, microscopic images of H&E stained normal livers and MYC-induced tumors. Scale bar = 100 μm. (B) SP analysis of LT2 normal liver cells and LT2-MYC hepatic tumor cells. (C) SP analysis of FVB/N normal liver cells and hepatic tumor cells induced by hydrodynamic transfection of AKT/RAS and MYC.

Fig. 2. SP cells are enriched for tumor-initiating potential

(A) Colony forming assay of unsorted LT2-MYC tumor cells (Total), sorted SP cells and non-SP cells performed in supplemented HpBGro serum-free media. (n=4), *p<0.00001 (B) Colony assays with SP and non-SP cells. (C) Primary allograft of LT2-MYC tumor cells. 10000 (n=4), 1000 (n=4) or 100 (n=4) unsorted LT2-Myc tumor cells (Total) and 10000 (n=4), 1000 (n=16) or 100 (n=12) sorted SP cells and 10000 (n=4), 1000 (n=16) or 100 (n=8) non-SP cells were seeded subcutaneously in NSG mice. Animals were monitored for tumors for 90 days post-injection. *p<0.02, **p<0.00001, ***p<0.003 (D) Secondary allograft of LT2-MYC tumors derived from SP cells. 100 (n=10) sorted SP cells and 100 (n=10) non-SP cells from LT2-Myc SP cell derived allograft tumors were subcutaneously seeded in NSG mice. Animals were monitored for tumors for 90 days post-injection. *p<0.000006 (E) Representative image of a NSG mouse seeded with 100 SP and 100 non-SP from LT2-Myc tumors in opposite flanks 60 days after injection.

Fig. 3. SP cells derived from liver tumors express markers of hepatic progenitor cells

(A) Q-PCR analysis of markers for hepatic progenitor cells (Cd44, Epcam and Bmi1) in unsorted (Total), sorted SP and non-SP LT2-MYC hepatic tumor cells, *p-value<0.002, **p-value<0.02, ***p-value <0.01 (B) Flow cytometry analysis for CD44 in SP and non-SP LT2-Myc hepatic tumor cells. Gating determined by verapamil control.

Fig. 4. SP cells derived from liver tumors have properties resembling those of hepatic progenitor cells

(A) SP analysis of LT2-MYC tumor cells grown in hepatocyte progenitor media for 7 days. SP gate was determined by loss of SP in verapamil treated controls. (B) SP analysis of LT2-Myc tumor cells grown on collagen-coated plates in mature hepatocyte differentiation media for 7 days. (C) Western blot analysis of whole cell lysate collected from LT2-MYC tumor cells grown on collagen-coated plates in mature hepatocyte differentiation ESP-Diff media with doxycycline (Doxy) (100 ng/ml) for 0, 4, 7 and 14 days. AFP is a marker for hepatic progenitors and C/EBPα is a marker for mature hepatocytes.

Fig. 5. MDR1 mediates formation of SP cells

(A) Q-PCR analysis of ABC transporter pump genes in unsorted (Total), sorted SP and non-SP LT2-MYC hepatic tumor cells and LT2 normal liver cells (Normal). Data are presented as fold-change relative to normal samples. *p-value<0.03, **p-value<0.002 (B) Western blot analysis of whole cell lysate from LT2 normal liver (N) and unsorted (T), sorted SP, non-SP LT2-Myc hepatic tumor cells and FVB/N wildtype kidney tissue (MRP1 and BCRP positive control). (C) SP analysis in the absence (top row) and presence (bottom row) of verapamil (50μM) of tumor cells from FVB/N Wt, Bcrp−/− and Mdr1a/1b−/− mice following MYC hydrodynamic transfection.

Fig. 6. MDR1 confers chemoresistance to MDR1-effluxed drugs but not BCRP-effluxed drugs in LT2-MYC SP cells

(A) MTT assay of LT2-MYC hepatic tumor cells following treatment for 72 hours with either Dox (10μM), PTX (0.1μM) or SN38 (0.1μM) (n=3–4). Cell viability is shown as a percentage of MTT conversion compared to 0.1% DMSO (PBS) treated samples. Parentheses denote drug transporters that preferentially efflux these drugs. (B) SP analysis of isolated LT2-MYC tumor cells treated overnight in vitro with 0.1% DMSO (PBS), PTX (2.5 nM) or Dox (2.5 nM). (C) TUNEL analysis of LT2-Myc tumors. LT2-MYC tumor bearing mice were treated with 0.1% DMSO (PBS) or PTX (200 μg) every 3 days and analyzed after 2 weeks of treatment. (D) SP analysis of LT2-MYC tumors following in vivo treatment with 0.1% DMSO (PBS) or PTX (200 μg). (E) LT2-MYC tumor cells were treated with either 0.1% DMSO (PBS) or PTX (200 μg). Following the treatment, 300 live cells from each sample (n=10) were subcutaneously seeded in NSG mice. Percentages of mice with tumors were evaluated up to 90 days post-injection.