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. 2011 Jul 7;2(7):e179.
doi: 10.1038/cddis.2011.61.

Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

M Saxena  1 , M A Stephens, H Pathak, A Rangarajan
Affiliations

Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

M Saxena et al. Cell Death Dis. .

Abstract

Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial-mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance.

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Figures

Figure 1
Figure 1
Breast epithelial cells have a heterogeneous expression of ABC transporters. (a) Representative graphs showing relative mRNA expression of ABC transporters in immortalized (MCF10A and HBL100), non-invasive (MCF7 and T47D), and invasive (MDAMB231 and MDAMB435) breast epithelial cell lines as analyzed by RT-PCR, n=3. (b) Representative graphs showing relative mRNA expression of ABC transporters in primary normal (NB), DCIS, and IDC breast samples as analyzed by RT-PCR. n=3 for NB, n=6 for DCIS, and n=3 for IDC samples
Figure 2
Figure 2
Dox treatment upregulates ABC transporter expression only in invasive cells. (a) Phase-contrast microscopic images taken after 1 week of untreated (UT) and doxorubicin-treated (Dox) MCF10A, MCF7, and MDAMB231 breast epithelial cell lines. Scale bar=100 μm. (b) Graphs represent relative mRNA expression of 16 ABC transporters after 1-week Dox treatment in MCF10A, MCF7, and MDAMB231 breast epithelial cell lines, n=3. Statistical significance for all the experiments was determined using ratio t-test. Error bars denote ±S.E.M. *P<0.05; **P<0.01; ***P<0.001. The insets represent immunoblot analysis of whole-cell lysates for the expression of three ABC transporters, viz, ABCB1, ABCC1, and ABCG2 in the absence or presence of Dox. (c) Phase-contrast microscopic images of primary breast tissue-derived cells in the presence (Dox) or absence (UT) of Dox for 7 days; scale bar=100 μm. (d) Representative graph showing relative mRNA expression of 16 ABC transporters after 1-week Dox treatment in primary normal- and tumor-derived breast epithelial cells, n=4. (e) Representative graph showing relative mRNA expression of ABC transporters in chemotherapy-treated patient-derived primary normal (CT-NB) and tumor (CT-CB) breast samples. n=2 for normal and n=3 for tumor samples
Figure 3
Figure 3
Dox treatment of invasive cells leads to the upregulation of EMT markers and invasiveness. (a) Graphs represent relative mRNA expression of EMT markers after 1-week Dox treatment in MCF10A, MCF7, and MDAMB231 cells, n=3. Error bars denote ±S.E.M. *P<0.05. (b) Immunoblot analysis of whole-cell lysates of MCF10A, MCF7, and MDAMB231 cells for the expression of EMT markers in the absence or presence of Dox. (c) Photomicrographs represent cell migration of untreated (UT) or doxorubicin-treated (Dox) MDAMB231 cells in wound healing assay at 0 and 24 h. Scale bar=500 μm. (d) Graph represents distance migrated by cells in the experiment (c) calculated as a difference of distance between the two edges of the wounds at 0 and 24 h measured in μm using the ProgRes capture software, n=8. Error bars denote ±S.E.M. **P<0.01. (e) Graph represents relative mRNA expression of MMP markers after 1 week of Dox treatment in MDAMB231 cells, n=3. Error bars denote ±S.E.M. *P<0.05; **P<0.01. (f) Graph represents invasion index calculated as ratio of percentage cell invasion of Dox-treated cells and -untreated control cells through Matrigel in Boyden chamber assay; n=2 for MCF10A and MCF7, and n=3 for MDAMB231. Error bars denote ±S.E.M. *P<0.05
Figure 4
Figure 4
EMT induction upregulates ABC transporter expression. Graphs represent relative mRNA expression of EMT markers (a) and ABC transporters (b) after 1-week treatment of MCF7 cells with TGFβ, n=4. Error bars denote ±S.E.M. *P<0.05; **P<0.01. Graphs represent relative mRNA expression of EMT markers (c) and ABC transporters (d) in MCF7 cells transiently overexpressing mTwist, n=4. Error bars denote ±S.E.M. *P<0.05; **P<0.01. (e) Phase-contrast images of parent HMLE cells and HMLE cells stably overexpressing mTwist (HMLE-mTwist); scale bar=100 μm. (f) Graph represents distance migrated by cells in wound migration assay calculated as a difference of distance between the two edges of the wounds at 0 and 24 h measured in μm using the ProgRes capture software, n=4. Error bars denote ±S.E.M. **P<0.01. (g) Graph represents cell invasion in arbitrary units (AU) calculated as a ratio of cells invaded through Matrigel to the total number of cells plated, n=3. Error bars denote ±S.E.M. **P<0.01. Graphs represent relative mRNA expression of EMT markers (h) and ABC transporters (i) in HMLE cells stably overexpressing mTwist, n=3. Error bars denote ±S.E.M. *P<0.05. (j) Dose–response curves of parent HMLE cells versus HMLE cells stably expressing mTwist/FOXC2/Snail treated with doxorubicin for 48 h. The dotted lines represent the IC50 values. (k) Graph represents relative mRNA expression of 16 ABC transporters in HMLE-mTwist cells after 1 week of Dox treatment, n=3. Error bars denote ±S.E.M. *P<0.05; **P<0.01; ***P<0.001
Figure 5
Figure 5
Reversal of EMT leads to increased drug sensitivity. (a) Photomicrographs of MDAMB231 cells transfected with control, Twist, or Zeb1 siRNA in the presence and absence of Dox treatment for 72 h. Scale bar=100 μm. Gel pictures depict RT-PCR analysis of EMT markers (b) and ABC transporters (c) in MDAMB231 cells transfected with control, Twist, or Zeb1 siRNA in the presence and absence of Dox treatment for 72 h. (d) Dose–response curves of MDAMB231 cells transfected with control, Twist, or Zeb1 siRNA, and subsequently treated with varying concentrations of Dox for 48 h. The dotted lines represent the IC50 values. Error bars denote ±S.E.M. *P<0.05; **P<0.01. (e) Graph represents percentage cell invasion through Matrigel in Boyden chamber assay for MDAMB231 cells transfected with control or Twist siRNA, and subsequently treated with Dox for 72 h, n=3. Error bars denote ±S.E.M. *P<0.05; ***P<0.001
Figure 6
Figure 6
EMT-inducing transcription factors regulate ABC transporter expression. (a) Graph represents luciferase assay undertaken in MCF7 cells to assess the ABCC5 promoter activity (ABCC5-Luc; firefly luciferase) on co-transfection with pRL-TK (Renilla luciferase) and pBp-empty vector/Snail/mTwist/FOXC2. After 48 h culture, firefly luciferase activity was measured and normalized to Renilla luciferase activity and depicted as relative luciferase units (RLU), n=3. Error bars denote ±S.E.M. *P<0.05; **P<0.01. (b) Schematic of E-box elements in 2.2 kb long promoter regions of ABCC4 and ABCC5 genes. (c) Gel picture represents PCR carried out on ChIP undertaken in MCF7 cells transiently overexpressing Flag-Twist using anti-Flag Ab. PCR was carried out with the eluted DNA fragments using primers depicted in (b). No antibody (No Ab) served as a negative control and rabbit IgG (Rab IgG) served as an isotype control. Control primers were designed ∼700 bp upstream of E-box elements in the ABCC5 promoter and served as a specificity control
Figure 7
Figure 7
A schematic representation of the effect of chemotherapy on invasive cancer cells. Chemotherapeutic treatment of a heterogeneous population of cancer cells kills majority of the epithelial-like cells, whereas the mesenchymal-like cells survive owing to a basal expression of ABC transporters. Drug-induced activation of self-renewal pathways (such as TGFβ) causes an increase in the expression of EMT transcription factors, mesenchymal morphology, migratory, and invasive potential of the surviving cells. These EMT factors can then upregulate the expression of a large number of ABC transporters simultaneously, resulting in the generation of cancer stem-like cells that are multidrug resistant
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