HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells - PubMed (original) (raw)

. 2012 Dec 15;18(24):6634-47.

doi: 10.1158/1078-0432.CCR-12-1436. Epub 2012 Oct 22.

Ming Fan, Demet Candas, Cheikh Menaa, Hsin-Chen Liu, Danupon Nantajit, Yunfei Wen, Kai Xiao, Angela Eldridge, Brett A Chromy, Shiyong Li, Douglas R Spitz, Kit S Lam, Max S Wicha, Jian Jian Li

Affiliations

HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells

Nadire Duru et al. Clin Cancer Res. 2012.

Abstract

Purpose: To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2(-/low) breast cancers.

Experimental design: HER2-expressing BCSCs (HER2(+)/CD44(+)/CD24(-/low)) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program.

Results: Compared with HER2-negative BCSCs, HER2(+)/CD44(+)/CD24(-/low) cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2(+)/CD44(+)/CD24(-/low) cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2-STAT3 network was identified.

Conclusion: This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2(-/low) breast cancer.

©2012 AACR.

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Conflict of interest statement

CONFLICT OF INTEREST: No potential conflicts of interests were disclosed

Figures

Figure 1

Figure 1

HER2 protein expression causes the aggressive growth in breast cancer cells with HER2−/low status. (A) HER2 protein expression was dose-dependently induced by IR in MDA-MB- 231 cells and in a radioresistant MCF7/C6 (C6) cells (8, 20). MDA-MB-231 were exposed to increased doses of irradiation (0–4 Gy). Twenty-four hours post-irradiation, cells were collected and tested for the expression of HER2. For MCF7/C6 cells, parental MCF7 cells (wt) and MCF7 cells stably transfected with HER2 gene (HER2) were used as negative and positive controls, respectively. (B) Evaluation of cell aggressiveness. Matrigel invasion assay: Cells were cultured in Matrigel and cells migrating through the transwell were counted to reflect cell’s aggressiveness (mean+SE; n=6; *p<0.05, **p<0.01; Depicted images of invaded cells are illustrated in Fig. S1C). The gap-filling capacity was calculated as the ratio of filled gaps at 96 h post scraping compared to the filled gap at 0 h (Additional data are shown in Fig. S1A). The tumor sphere formation was evaluated as the number of tumor spheres formed per dish (mean+SE; n = 6; **p<0.01; Images of tumor sphere formation are illustrated in Fig. S1D). Clonogenic survival was evaluated as the percentage of cells seeded that were able to form colonies 14 days after irradiation (mean+SE; n = 3; **p<0.01). (C) The radioresistant phenotype of MCF7/C6 (C6) cells was compared with MCF7/HER2 (HER2) and the parental MCF7 cells after treatment with 5 Gy IR by measuring apoptosis, clonogenic survival, and gap filling rates (mean+SE; n=3; **p<0.01). (D) Quantification of BCSCs with the feature of HER2+/CD44+/CD24−/low in the surviving fraction of MCF7 cells (left) or xenograft tumors (right) treated with 5 × 2 Gy IR. Cell suspensions from control (sham) or irradiated cells or tumors were sorted by FACS with conjugated antibodies (APC for HER2, PE for CD44, and FITC for CD24; n = 3 from separate sorting, *p<0.05, **p<0.01; additional FACS data are shown in Fig. S3 and Table S1).

Figure 2

Figure 2

Characterization of the HER2+/CD44+/CD24−/low BCSCs within MCF7/C6 cell lines. (A) CD44+/CD24−/low cells were sorted from 2.5 × 107 MCF/C6 cells with CD44 and CD24 antibodies (left panel), from which the cells were further sorted with HER2 antibody (right panel). The orange box in the left panel indicates the CD44+/CD24−/low fraction used for sorting HER2+/CD44+/CD24−/low (yellow box), HER2−/CD44+/CD24−/low cells (red box) and a global HER2−/CD44+/CD24−/low population (blue box with a broader gate). (B) Percentages of CD44+/CD24−/low and HER2+/CD44+/CD24−/low cells derived from MCF7/C6 cells (left panel, also shown in Table S2). Western blot analysis of HER2 and CD44 protein expression in sorted HER2+/CD44+/CD24−/low and HER2−/CD44+/CD24−/low cells (right panel). (C) Analysis of ALDH activity in HER2−/CD44+/CD24−/low versus HER2+/CD44+/CD24−/low cells via flow cytometry using Aldefluor staining. As negative control, cells were incubated with ALDH inhibitor DEAB. (D) Quantification of the ALDH expression HER2+/CD44+/CD24−/low compared to HER2−/CD44+/CD24−/low BCSCs analysis in C.

Figure 3

Figure 3

Enhanced radiation resistance and aggressiveness in HER2+/CD44+/CD24−/low BCSCs. (A) Mitochondrial membrane potential (left panel) and apoptosis (right panel) were measured in HER2+/CD44+/CD24−/low and HER2−/CD44+/CD24−/Low BCSCs treated with or without 5 Gy IR for 24 h. Data represents mean+ SE of 3 independent experiments and statistical significance was evaluated as *p<0.05; **p<0.01. (B) Enhanced radioresistance in HER2+/CD44+/CD24−/low cells treated with sham (0 Gy) or 5 Gy of IR is tested by gap filling and clonogenic survival assays. The gap-filling rate (left panel) was calculated as the ratio of the filled gap at 96 h compared with 0 h. The clonogenic survival (right panel) of HER2+/CD44+/CD24−/Low BCSCs was evaluated by counting the number of colony formation (more than 50 cells) 14th day after cells were treated with 5 Gy of IR (n = 6 and **p<0.01; Additional data are shown in Fig. S5). (C) The aggressiveness of HER2−/CD44+/CD24−/low (HER2−) and HER2+/CD44+/CD24−/low (HER2+) BCSCs was further analyzed by Matrigel invasion assay (left panel), tumor sphere formation (middle panel, n = 6; **p<0.01; scale bar = 50 μm) and in vivo tumorigenesis (right panel). Tumor numbers and volumes were measured in NOD/SCID mice two weeks after the inoculation of 500 cells from each BCSC population in two opposite site (HER+ left side and HER- right side) of the animal as depicted by arrows.

Figure 4

Figure 4

Inhibition of HER2 blocks the aggressiveness and radioresistant phenotypes of HER2+/CD44+/CD24−/low BCSCs. (A) Left panel represents depicted images of Matrigel invasion assays of HER2+/CD44+/CD24−/low BCSCs at 72 h after 5 Gy IR and treatment with scrambled or specific HER2 siRNA. Middle panel, depicts the specific capacity of HER2 siRNA to reduce HER2 expression in HER2+ breast cancer stem cells. Right panel represents quantification of the number of cells invading the membrane per transwell. Data represents mean+SE of n = 6 and statistical analysis was performed with *p<0.05, **p<0.01). (B) Represents the gap filling values determined at different time points post cell’s injury (0 to 96 h) in sham (0 Gy) and irradiated cells (5Gy) and in cells treated with specific or scrambled siRNA against HER2. Right panel represent mean +SE of the gap filling rate calculated from n = 6 independent experiments; **p<0.01). (C, D) The effect of targeting HER2 signaling pathway on aggressiveness and radiosensitivity of BCSCs. HER2+/CD44+/CD24−/low were treated either with siRNA (C; n = 3; *p<0.05; **p<0.01) or with anti- HER2 antibody, Herceptin, (D; 10 μg/ml for 5 days, n = 3; **p<0.01). The clonogenic sensitivity to IR was evaluated following sham or 5 Gy IR (mean+SE; n=3).

Figure 5

Figure 5

Increased number of HER2+/CD44+ cells in recurrent breast cancer. (A) HER2 expression status was assessed in breast cancer samples that were diagnosed as primary, recurrent or unknown status by FISH and IHC. Blue: HER2 + (IHC 2+ and 3+) or amplified (FISH, HER2: centromere 17 signal ratio >2.2); Green: HER2-(IHC 0) or non-amplified (FISH, HER2:centromere 17 signal ratio <1.8); Red: HER2 equivocal (IHC 1+ and FISH, HER2:centromere 17 signal ratio =1.8 – 2.2). (B) Data of immunohistochemistry analysis for the expression of HER2 and CD44 in tumors harvested from breast cancer patients. Positive cells are depicted with arrows. HER2−/CD44− tumor was assigned if no HER2+/CD44+ cells were detected. Positive tumors were assigned if at least one cluster of HER2+/CD44+ was detected (Additional staining results are shown in Fig. S6).

Figure 6

Figure 6

HER2 and STAT3 crosstalk in HER2+/CD44+/CD24−/low BCSCs. (A) HER2 and STAT3 protein expression levels were further determined by Western blot in the HER2+/CD44+/CD24−/low and HER2−/CD44+/CD24−/low BCSCs. (B) Increased STAT3 transcriptional activity in HER2+/CD44+/CD24−/low compared to HER2−/CD44+/CD24−/low BCSCs. STAT3 luciferase reporters (52) were transfected in HER2+/CD44+/CD24−/low and HER2−/CD44+/CD24−/low cells and luciferase activity was measured 24 h after transfection (n = 3; **p < 0.01). (C) Co-localization of HER2 and STAT3 expression in radio-resistant cancer cells and (D) HER2+ human breast specimen. (E) A putative HER2-STAT3 crosstalk signaling networks for the aggressiveness of HER2+/CD44+/CD24−/low BCSCs. For the interaction between HER2 and STAT3 signals, a cluster of factors searched from the database of MetaCore™ Version 6.9, was identified to interact with both STAT3 and HER2. This network causing a crosstalk between HER2 and STAT3 may play a critical role for the overall aggressiveness of the HER2 expressing breast cancer stem cells in breast tumors with HER2−/low status.

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