The mammary glands of Cyclin D1-deficient females in an FVB background exhibit extensive alveologenesis during pregnancy and consequently must possess alveolar progenitors that, as we have reported previously (15
), are the main targets for ErbB2-induced neoplastic transformation. Although lack of Cyclin D1 extended the tumor-free survival in our study, Cyclin D1 deficiency did not prevent the onset of mammary cancer as reported previously (10
). Following neoplastic transformation, Cyclin D1-deficient mammary tumors exhibited the same sporadic occurrence, growth, and histopathological features compared to cancers that arose in females expressing Cyclin D1. The difference in the outcome of our study from that of others can be explained by the effects of diverse strain backgrounds. The previous reports were based on the maintenance of the MMTV-neu transgene in a predominantly 129/C57 mixed genetic background. While Cyclin D1-deficient females carrying C57 alleles lack alveolar progenitors and therefore the cancer-initiating cell type, there is also compelling evidence from a study by Rowes and colleagues (38
) that suggests that the genetic background has a profound impact on the tumor latency in MMTV-neu mice. While the underlying mechanism for this phenomenon remains unknown, it is evident that genetic studies related to MMTV-neu-induced mammary tumorigenesis should be performed in the FVB strain.
A delay or lack of tumor formation in a knockout mouse expressing an oncogene may not be an appropriate indicator for whether the targeted ablation of a gene or its encoded protein is also relevant for therapeutic approaches in humans and animals (41
). Cancer cells that evolve though selective mechanisms frequently shift signaling networks and employ alternative pathways to optimize growth and survival as demonstrated recently for the Jak/Stat pathway (28
). A suitable experimental design to assess the importance of a protein during cancer progression is to repress its expression within the cancer cells of an established neoplasm. Based on this concept, we have generated a genetic model that allows the targeted downregulation of Cyclin D1 in progressing ErbB2-positive mammary cancers. Using this tumor model, we demonstrated that this cell cycle regulator is not essential for the proliferation of cancer cells, and consequently Cyclin D1 may not be a primary candidate target to treat ErbB2-associated breast cancer as suggested previously.
While Cyclin D1 has long been recognized as an oncogene in breast carcinogenesis, considerably fewer studies included an examination of the expression and functionality of the other two D-type cyclins. The first immunohistochemical analysis of Cyclin D3 expression in human malignancies by Bartkova et al. (42
) revealed an overabundance of this particular cell cycle regulator in breast cancer specimens. A synchronous upregulation of Cyclin D1 and D3 in a subset of human breast cancers was also reported by Russell and colleagues (43
), and the authors proposed that this was, in part, a consequence of defective proteolysis. A subsequent study by the same research team demonstrated that overexpression of Cyclin D3 is sufficient to induce the development of squamous carcinomas in the mammary gland of transgenic mice (44
). Using western blot analysis and quantitative immunofluorescence (i.e., two independent assays with different antibodies) on breast cancer cell lines and primary tumor specimens, we show in this report that the extent of Cyclin D3 upregulation in human breast cancer cells often exceeds that of Cyclin D1. In particular, ErbB2-positive cases that are known to have a poor prognosis express significantly more Cyclin D3 compared to Cyclin D1. Our findings are in line with an empirical study by Wong et al.
) that shows that Cyclin D3 was more abundant than Cyclin D1 in high-grade breast cancers.
In this study, we provide several lines of evidence that the expression of D-type cyclins is concordantly regulated in mammary cancer cells, and only the combined inhibition of Cyclin D1 and D3 had a profound impact on cancer cell proliferation. We did not observe a compensatory upregulation or a gain-of-function of Cyclin E that was sufficient to bypass the importance of the D-type cyclins as proposed previously for ErbB2-associated mammary cancer (46
) and normal MECs expressing exogenous ERα (47
). Collectively, our findings suggest that targeting the combined functions of Cyclin D1 and D3 might be a suitable strategy for breast cancer therapy. These D-type cyclins regulate both Cdk4 and Cdk6, and it has been shown recently that a highly specific and well-tolerated Cdk4/6 dual inhibitor (PD-0332991) can effectively block the proliferation of selected ERα-positive and ERα-negative breast cancer cells that have normal Rb function (48
). In the light of these encouraging findings, our study suggests extending the use of this Cdk4/6 inhibitor or similar agents to treat ErbB2-positive breast cancer cases.