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. 2012 Nov 1;11(21):4020-32.
doi: 10.4161/cc.22225. Epub 2012 Sep 19.

Epithelial-to-mesenchymal transition (EMT) confers primary resistance to trastuzumab (Herceptin)

Cristina Oliveras-Ferraros  1 , Bruna Corominas-Faja, Sílvia Cufí, Alejandro Vazquez-Martin, Begoña Martin-Castillo, Juan Manuel Iglesias, Eugeni López-Bonet, Ángel G Martin, Javier A Menendez
Affiliations

Epithelial-to-mesenchymal transition (EMT) confers primary resistance to trastuzumab (Herceptin)

Cristina Oliveras-Ferraros et al. Cell Cycle. .

Abstract

The rate of inherent resistance to single-agent trastuzumab in HER2-overexpressing metastatic breast carcinomas is impressive at above 70%. Unfortunately, little is known regarding the distinctive genetic signatures that could predict trastuzumab refractoriness ab initio. The epithelial-to-mesenchymal transition (EMT) molecular features, HER2 expression status and primary responses to trastuzumab were explored in the public Lawrence Berkeley Laboratory (LBL) Breast Cancer Collection. Lentivirus-delivered small hairpin RNAs were employed to reduce specifically and stably the expression of EMT transcription factors in trastuzumab-refractory basal/HER2+ cells. Cell proliferation assays and pre-clinical nude mice xenograft-based studies were performed to assess the contribution of specific EMT transcription factors to inherent trastuzumab resistance. Primary sensitivity to trastuzumab was restricted to the SLUG/SNAIL2-negative subset of luminal/HER2+ cell lines, whereas all of the SLUG/SNAIL2-positive basal/HER2+ cell lines exhibited an inherent resistance to trastuzumab. The specific knockdown of SLUG/SNAIL2 suppressed the stem-related CD44+CD24(-/low) mesenchymal immunophenotype by transcriptionally upregulating the luminal epithelial marker CD24 in basal/HER2+ cells. Basal/HER2+ cells gained sensitivity to the growth-inhibitory effects of trastuzumab following SLUG/SNAIL2 gene depletion, which induced the expression of the mesenchymal-to-epithelial transition (MET) genes involved in promoting an epithelial phenotype. The isolation of CD44+CD24(-/low) mesenchymal cells by magnetic-activated cell sorting (MACS) confirmed their intrinsic unresponsiveness to trastuzumab. A reduction in tumor growth and dramatic gain in sensitivity to trastuzumab in vivo were confirmed when the SLUG/SNAIL2 knockdown basal/HER2+ cells were injected into nude mice. HER2 overexpression in a basal, rather than in a luminal molecular background, results in a basal/HER2+ breast cancer subtype that is intrinsically resistant to trastuzumab. EMT transcription factors might induce an enhanced phenotypic plasticity that would allow basal/HER2+ breast cancer cells to "enter" into and "exit" dynamically from trastuzumab-responsive stem cell-like states. The systematic determination of SLUG/SNAIL2 as a stem/CD44+CD24(-/low) cell-associated protein may improve the therapeutic management of HER2+ breast carcinomas.

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Figures

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Figure 1. Association of the clinical subtypes of breast cancer cell lines with the primary response to the anti-HER2 monoclonal antibody trastuzumab. The relation between the morphological subgroups of breast cancer cell lines (luminal, basal-A, basal-B/mesenchymal), and breast tumor subtypes (luminal A, luminal B, HER2-enriched, basal-like, claudin-low) was evaluated by assessing the status of HER2 gene amplification, the primary response to trastuzumab and the expression status of SLUG/SNAIL2 as discriminators. As a group, the trastuzumab-resistant basal/HER2+ cell lines overexpressed the EMT transcriptional factor SLUG/SNAIL2, whereas trastuzumab-sensitive luminal/HER2+ cell lines underexpressed SLUG/SNAIL2.
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Figure 2. Relative enrichment of EMT features in trastuzumab-resistant basal/HER2+ JIMT1 breast cancer cells. Figure shows the relative expression of EMT-associated genes in basal/HER2+ JIMT1, basal A MDA-MB-468 and basal B MDA-MB-231 breast cancer cell vs. luminal/HER2+ SKBR3 breast cancer cells using the human epithelial-to-mesenchymal (EMT) RT2 Profiler PCR Array (PAHS-090, 96-well format) as per the manufacturer’s instructions (SABiosciences).
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Figure 3. Impact of shRNA-driven genetic ablation of EMT transcription factors on CD44+CD24-/low cell surface markers in basal/HER2+ JIMT1 cells. Left parts: Provided are representative flow cytometry dot plots of CD44 and CD24 expression of JIMT1 cells engineered to stably exhibit knocked-down expression of SLUG/SNAIL2, TWIST1 or ZEB1. Right parts: The average percentage of CD44+CD24-/low (red) and CD44+CD24+ (blue) cells determined by flow cytometry was calculated from three independent experiments.
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Figure 4. Impact of shRNA-driven genetic ablation of EMT transcription factors on the epithelial-to-mesenchymal (EMT) genetic program in basal/HER2+ JIMT1 cells. Total RNA from shControl-JIMT1, SLUG/SNAIL2 KD-JIMT1, TWIST1 KD-JIMT1, ZEB1 KD-JIMT1 cells was characterized in technical triplicates using a customized PCR array as described in the Materials and Methods section. Figures show representative scatter plots of the difference (≥ 2-fold; green and red symbols indicate downregulation and upregulation, respectively vs. basal expression levels in shControl-transduced JIMT1 cells) in relative transcript abundance of VIM, E-cadherin, N-cadherin, fibronectin, SNAIL1, SLUG/SNAIL2, SNAIL3, TWIST1, ZEB1 and ZEB2. Grey symbols denote fold-change results that need to be validated with a sufficient number of biological replicates—i.e., fold-change results may have greater variations if p value > 0.05, or the p value for the fold-change is either unavailable or relatively high (p > 0.05)—or they are interpretable because gene’s average threshold cycles was either not determined or greater than the defined cut-off value (default 35) in both samples. KD, knockdown
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Figure 5. Impact of the shRNA-driven genetic ablation of EMT transcription factors on trastuzumab-induced inhibition of breast cancer cell proliferation. shControl-JIMT1, SLUG/SNAIL2 KD-JIMT1, TWIST1 KD-JIMT1 and ZEB1 KD-JIMT1 cells were plated in 24-well plates at a density of 10,000 cells/well and cultured with regular media in the absence or presence of 100 μg/mL trastuzumab. The data presented are the means of number cells × 104/well and 95% confidence intervals (bars) from three independent experiments made in duplicate and obtained after 0, 3, 6 and 8 d. KD, knockdown; Tzb, trastuzumab.
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Figure 6. Immunohistochemical characterization of CD44+CD24-/low mesenchymal cells sorted from trastuzumab-refractory, basal/HER2+ JIMT1 cells. Trastuzumab-refractory, basal/HER2+ JIMT1 cells were sorted into CD44+CD24-/low-depleted and CD44+CD24-/low-enriched populations (top panels) and then stained for CD44, CD24 and the EMT marker VIM immediately following sorting (bottom panels). Enrichment of target cells by magnetic MicroBeads (MACS® Technology) was performed according to the manufacturer’s protocol. Representative immunofluorescent stainings of CD44 (red), CD24 (green), VIM (green) and Hoechst 33258 (blue) are presented. CD44+CD24-/low mesenchymal cells were found to have a high nucleo/cytoplasmic (N/C) ratio and a greater expression of VIM.
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Figure 7. Impact of the shRNA-driven genetic ablation of SLUG/SNAIL2 on the efficacy of trastuzumab in vitro and in vivo. Top panels: Changes in the cell viability of trastuzumab-resistant JIMT1 cell populations depleted or enriched for CD44+CD24-/low mesenchymal cells. The metabolic status of unsorted JIMT1 parental cells, CD44+CD24-/low-depleted, and CD44+CD24-/low-enriched JIMT1 cells treated with graded concentrations of trastuzumab before and after SLUG/SNAIL2 knockdown was evaluated using MTT-based cell viability assays and constructing dose-response graphs as % of untreated cells (untreated control cells = 100% cell viability). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Bottom panels: Changes in the tumor volume of shControl-JIMT1 and SLUG/SNAIL2 KD-JIMT1 xenografts grown in immunodeficient mice treated with trastuzumab (5 mg/kg/week).
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Figure 8. Mammosphere morphology of basal/HER2+ JIMT1 cells in response to the specific knockdown of SLUG/SNAIL2. The impact of shRNA-driven genetic ablation of SLUG/SNAIL2, TWIST1 and ZEB1 in mammosphere morphology was determined after seeding JIMT1 parental cells and SLUG/SNAIL2 KD-JIMT1, TWIST1 KD-JIMT1 and ZEB1 KD-JIMT1 cells in ultralow attachment plates with mammosphere medium for 5–7 d. Immunostaining of E-cadherin was performed on established spheres to visually evaluate whether the knockdown of EMT transcriptional drivers may result in disrupted mammosphere morphology. Genetic ablation of EMT drivers by lentivirus-delivered shRNAs did not affect mammosphere morphology of basal/HER2+ JIMT1 cells. KD, knockdown
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