Tumor-Initiating Cells of HER2-Positive Carcinoma Cell Lines Express the Highest Oncoprotein Levels and Are Sensitive to Trastuzumab

Abstract

Purpose: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. Experimental Design: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1signaling inhibitor (GSI) or Notch1small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. Results: In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following g-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. Conclusions: Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.

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

“…Magnifico et al 16 further expanded this notion to demonstrate that breast CSCs are exquisitely sensitive to trastuzumab, because they appear to express the highest levels of the HER2 protein, regardless of their HER2 gene amplification status.…”

“…In this scenario, the SLUG/SNAIL2-driven CD44 + CD24 -/low mesenchymal immunophenotype in the HER2 gene-amplified genomic background may induce an enhanced phenotypic plasticity that would allow the basal/HER2 + breast cancer cells to "enter" into and "exit" dynamically from HER2-driven stem cell-like states, which are primarily expected to be sensitive to trastuzumab. 1,16 It is well-established that some members of the SNAIL family (i.e., SLUG/SNAIL2) and TWIST can confer an EMT phenotype to breast epithelial cells, a phenomenon that generally correlates with the cells changing their phenotype from CD44 -CD24 + to CD44 + CD24 -/low . [58][59][60][61][62] We now confirm the possibility that the EMT drivers SLUG/SNAIL2 and TWIST can also participate in the conversion of CD44 + CD24 + basal epithelial cells into CD44 + CD24 -/low mesenchymal cells.…”

Transcriptional upregulation of HER2 expression in the absence of HER2 gene amplification results in cetuximab resistance that is reversed by trastuzumab treatment

“…The benefits of adjuvant trastuzumab may not be limited to patients with HER2 gene amplification (13,14), and trastuzumab efficiently triggers receptor-enhanced chemosensitivity (REC) when combined with chemotherapy in cell lines with low-to-intermediate HER2 expression, i.e., without HER2 overexpression (15,16). Moreover, trastuzumab may work by targeting cells with tumor-propagating properties; such cells, notably, are enriched in Ctx-resistant pooled populations of A431 cells (11), which have increased levels of HER2 protein compared with the bulk cell population, in which HER2 gene amplification is unmodified (17)(18)(19). Although this is an in vitro study, if our current data were to be validated with clinical samples, the threshold values for HER2 positivity may need to be redefined for Ctx-refractory patients with wild-type KRAS that might benefit from receiving a Ctx/trastuzumab combination.…”

Ductal carcinoma in situ of the breast: the importance of morphologic and molecular interactions

“…HER2 promotes carcinogenesis by maintaining and increasing cancer stem cells [30]. Interestingly, stem cells that overexpress HER2 are more sensitive to trastuzumab and lapatinib [31]. In some studies, the expression of HER2/neu in DCIS has been linked to recurrence following surgical excision without radiation therapy [7,32].…”