The “cancer stem cell hypothesis” proposes that cancers originate in tissue stem or progenitor cells through disregulation of the normally tightly regulated process of self-renewal. As a consequence cancers are composed of a cellular hierarchy with a limited number of “cancer stem cells” capable of self-renewal driving the malignant phenotype and differentiation generating tumor heterogeneity (Korkaya and Wicha, 2007
). A frequent genetic alteration in human breast cancer is the amplification of the HER2 gene (Slamon et al., 1987
). Although HER2 overexpression has been demonstrated to affect tumor growth, invasion and metastasis, the mechanisms involved remain undefined. We provide evidence that the effects of HER2 on mammary carcinogenesis, tumorigenicity and invasion result from effects of this signaling pathway on the cancer stem cell population. Overexpression of HER2 in normal mammary epithelial cells increases the stem/progenitor cell population as demonstrated by increased ALDH activity and mammosphere formation in vitro
and generation of hyperplastic lesions in NOD/SCID mice. In contrast, Aldefluor-negative HER2 expressing cells neither formed mammospheres nor produced outgrowths in humanized NOD-SCID mice.
In women, HER2 overexpression frequently occurs in premalignant lesions including atypical ductal hyperplasia (ADH) and ductal carcinoma in situ
(DCIS), suggesting that dysregulation of HER2 signaling plays a role in tumor initiation. Our findings suggest that HER2 amplification may lead to an expansion of mammary stem cells, providing targets for further carcinogenic events. This model could account for the demonstrated clonality of ADH and DCIS (Allred et al., 1994
; Park et al., 2006
; Xu et al., 2002
). HER2 overexpression may also affect the behavior of mammary carcinomas by expanding their “cancer stem cell” populations. We show that overexpression of HER2 in a series of human breast cancer cell lines increases the percentage of cells with ALDH activity. The functional significance of this was demonstrated by the ability of Aldefluor-positive/HER2 cells to form tumors which could be serially passaged in NOD/SCID mice. Despite equivalent levels of HER2 expression in Aldefluor-positive and negative cell populations, Aldeflour-negative cells failed to form tumors even with 100-fold more cells. HER2 overexpression also increased the tumorigenicity within the Aldefluor-positive population. These experiments suggest that HER2 increases the cancer stem cell population as well as increasing tumorigenicity within this population. In contrast, HER2 overexpression does not affect the phenotype of more differentiated Aldefluor-negative cells.
A number of genes and signaling pathways have been identified which regulate the self-renewal of stem cells in a variety of developmental systems. These include Oct3/4 and genes involved in the Notch and Hedgehog signaling pathways (Dontu et al., 2004
; Liu et al., 2006
; Tokuzawa et al., 2003
). We demonstrate that these genes show increased expression in Aldefluor-positive as compared to Aldefluor-negative Sum159 cells. Furthermore, Aldefluor-positive HER2-positive cells showed increased expression of these stem cell genes compared to Aldefluor-positive DsRed control cells. This suggests that in addition to increasing the Aldefluor-positive population, HER2 overexpression may also drive expression of stem cell regulatory genes within this population. HER2 has been shown to signal through the PI3-K Akt pathway generating a multidrug resistance phenotype (Knuefermann et al., 2003
). We found that the Aldefluor-positive cell population displayed significantly higher Akt phosphorylation than Aldefluor-negative cells. AKT phosphorylation was further increased by HER2 overexpression in this cell population.
A recent report suggested that the subpopulation of breast cancer cells, previously characterized by the stem cell phenotype CD44+/CD24− (Al-Hajj et al., 2003
) show higher levels of proinvasive genes and exhibit increased invasive properties (Sheridan et al., 2006
). We demonstrate here that HER2 overexpression increases the invasiveness of both MCF7 and SUM159 cells. Furthermore, the Aldefluor-positive populations are significantly more invasive than the Aldefluor-negative populations. These results suggest that HER2 overexpression may have a direct effect on tumor invasion as well as an indirect effect resulting from increases in the invasive cancer stem cell population.
In the metastatic setting, trastuzumab increases the effectiveness of chemotherapy resulting in an increased response rate and duration of response (Burstein et al., 2001
). Recent evidence suggests that CD44+/CD24− breast cancer stem cells may be relatively resistant to both chemotherapy and radiation. In contrast to the resistance of cancer stem cells to these conventional treatments, our results suggest that the effectiveness of trastuzumab may be directly related to its effects on the cancer stem cell population. A recent report demonstrating that the HER2/EGFR inhibitor, Lapatinib when used in neoadjuvant setting decreases the cancer stem cell component supports the clinical relevance of our findings (Li et al., 2008
Despite the significant clinical benefits of trastuzumab, many patients treated in the adjuvant setting still relapse and most patients with advanced disease become trastuzumab-resistant within one year (Paik et al., 1990
; Slamon et al., 1987
). The development of secondary mutations such as deletion of the tumor suppressor PTEN may contribute to trastuzumab resistance (Nagata et al., 2004
; Saal et al., 2007
). We demonstrate that treatment of SUM159-HER2 cells with trastuzumab results in an 80% reduction of the Aldefluor-positive cell population. The effect of trastuzumab on Sum159-HER2 cells was associated with decreased expression of phospho-HER2 and phospho-Akt. In contrast to its effect on SUM159-HER2 cells, trastuzumab had no measurable effect on phospho-HER2, phopho-Akt, or on the proportion of the Aldefluor-positivity in two trastuzumab resistant cell lines. However, the PI3-K inhibitor LY294002 reduced the Aldefluor-positive population in both trastuzumab sensitive and resistant cell lines. These findings are consistent with previous reports showing increased Akt activity in trastuzumab-resistant cell lines (Kucab et al., 2005
; Tanner et al., 2004
) and suggest that the PI3-K/Akt pathway plays an important role in mediating the effects of HER2 signaling. It is also consistent with recent reports suggesting a role for Akt signaling in stem cell self-renewal (Welham et al., 2007
) and in mediating treatment resistance (Ma et al., 2007
; Saal et al., 2007
). Most recently, Berns et al. identified the PI3K pathway as a major determinant of trastuzumab resistance in human breast tumors using a functional genomic approach (Berns et al., 2007
In conclusion, our results suggest that the effects of HER2 amplification on mammary carcinogenesis, tumorigenicity and invasion result from effects of this signaling pathway on mammary cancer stem cells. These effects are due to the ability of HER2 to expand the stem/progenitor cell population as well as increasing the tumorigenicity and invasiveness of this population. Carcinogenesis may be initiated by expansion of stem cell pools which provide targets for further carcinogenic events. Increased HER2 expression in breast cancers, in turn, may increase the cancer stem cell component driving tumorigenesis invasion and metastasis. An elucidation of the molecular mechanisms by which HER2 signaling drives the stem cell pool may lead to further insights into the behavior of normal and malignant mammary stem cells. In addition, identification of components of these pathways may lead to new targets for cancer prevention and therapy.