Cyclin-Dependent Kinase 4 Expression Is Essential for Neu-Induced Breast Tumorigenesis

A key response to growth factors in many cell types is the activation of cyclin-dependent kinase (Cdk) 4 or Cdk6 by members of the cyclin D family (D1, D2, and D3). D-type cyclins are expressed at low levels in a variety of quiescent cell types and their expression is stimulated by growth factors and mitogens (1–5). Approximately 50% of human mammary carcinomas express abnormally high levels of cyclin D1 (6–10), which is maintained throughout subsequent stages of breast cancer progression from in situ carcinoma to invasive carcinomas (9, 11, 12). Consistent with the oncogenic role of cyclin D1 in mammary epithelium, transgenic mice overexpressing cyclin D1 in their breast tissue have been found to develop mammary adenocarcinomas (13). Furthermore, loss of cyclin D1 was found to affect breast development (14, 15). More importantly, cyclin D1 null mutant mice were found to be resistant to breast cancers induced by the neu and ras oncogenes but remained fully sensitive to other oncogenic pathways driven by c-Myc or Wnt-1 (16). A requirement for D-type cyclins in cellular transformation in vitro has also been shown using triple cyclin D knockout mouse embryonic fibroblasts, which are resistant to transformation by c-Myc or Ras in combination with dn-p53, E1A, or c-Myc (17). Similarly, Cdk4 null mouse embryonic fibroblasts have been shown to be refractory to transformation by Ras and dn-p53 and, consistent with these data, the hyperactive Cdk4R24C allele cooperates with single oncogenes to transform mouse embryonic fibroblasts in vitro (18, 19). Taken together, these results suggest that the activity of D-type cyclin/Cdk4 complexes is required for fibroblast transformation. However, it has also been suggested that the oncogenic function of cyclin D1 is independent of its ability to activate Cdks and is perhaps linked to the direct effects of cyclin D1 in controlling the expression of a subset of genes that are co-up-regulated in human tumors with deregulated cyclin D1 (20).

Thus, whereas a role for cyclin D1 in breast cancer is well established, it is not known whether the oncogenic function of cyclin D1 requires Cdk4. To understand the role of Cdk4 in vivo, we have targeted the mouse Cdk4 locus by homologous recombination in embryonic stem cells and generated a strain of mice that does not express Cdk4 [Cdk4(neo/neo); ref. 21]. Homozygous Cdk4(neo/neo) null mutant mice are viable and were found to be very resistant to carcinogen-induced cancers (data not shown). In this communication, we show that loss of Cdk4 expression results in poor mammary gland development that is characterized by impaired ductal branching. In addition, we show that Cdk4 expression is essential for neu-induced breast tumor development; on the other hand, it is dispensable for wnt-induced breast tumor development.

Generation of Cdk4(neo/neo)/mouse mammary tumor virus transgenic mice. To generate compound mice that express neu and wnt-1 oncogenes in a Cdk4 null background, Cdk4(neo/+) mice were mated with mouse mammary tumor virus (MMTV)-neu and MMTV-wnt-1 transgenic mice to generate Cdk4(neo/+)/MMTV-neu and Cdk4(neo/+)/MMTV-wnt-1 mice, respectively. These mice were then intracrossed to generate Cdk4(neo/neo)/MMTV-neu and Cdk4(neo/neo)/MMTV-wnt-1 transgenic mice.

Whole-mount and histopathologic analysis of mammary glands. The fourth inguinal mammary glands were dissected, spread onto a glass slide, and fixed with a mixture (1:3) of glacial acetic acid/ethanol, hydrated, stained with 0.2% carmine and 0.5% AlK(SO₄)₂, dehydrated in graded solutions of ethanol, and cleared in toluene and methyl salicylate as described previously (14). Carmine-stained or formalin-fixed mammary glands were also routinely processed for paraffin embedding and were stained with H&E.

Protein analysis. Mammary glands or tumors were homogenized in TNE lysis buffer and lysates were cleared by centrifugation. Protein, 50 to 100 μg, was resolved by SDS-PAGE and was transferred to nitrocellulose membranes. Immunoblots were probed with antibodies against HER2/ErbB2 (Cell Signaling Technology, Beverly, MA), Cdk4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Cdk6 (NeoMarkers, Fremont, CA), Cdk2 (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abcam, Cambridge, MA), retinoblastoma (Rb; BD Biosciences, San Diego, CA), and phosphorylated Rb (pRb; Ser⁷⁸⁰; Cell Signaling Technology).

Cdk4 is required for proper development of mammary epithelium. To gain an insight into the role of Cdk4 in breast development, we first examined the status of mammary epithelium in wild-type [Cdk4(+/+)] and Cdk4-deficient [Cdk4(neo/neo)] mice. Examination of H&E-stained mammary gland whole mounts derived from virgin female mammary glands at 14 to 17 weeks revealed striking differences in the extent of mammary gland ductal outgrowth in the two sets of mice (Fig. 1A and C). In Cdk4(neo/neo) mice, both ductal outgrowth and branching morphogenesis was considerably reduced when compared with their wild-type counterparts. In addition, an examination of the longitudinal sections of the mammary tissue sections also showed a distinctive reduction in the number of mammary ducts and a complete absence of alveoli (Fig. 1B and D). These observations suggest that loss of Cdk4 expression in breast epithelium results in a diminution of mammary gland ductal branching where alveolar segments were markedly fewer in number compared with the wild-type mammary gland.

Effect of loss of Cdk4 expression on neu- and wnt-mediated breast tumorigenesis. To study the role of Cdk4 in neu- and wnt-induced breast tumorigenesis, Cdk4(neo/neo) mice were bred with MMTV-neu and MMTV-wnt transgenic mice to generate Cdk4(neo/neo):MMTV-neu, and Cdk4(neo/neo):MMTV-wnt mice, respectively (Fig. 2A). Whole-mount and histopathologic sections of the mammary glands derived from virgin adult mice (∼14 weeks) from these different crosses (Fig. 2B and F) showed that Cdk4(+/+):MMTV-neu mice exhibit proliferative disturbances in the mammary epithelium as evidenced by the appearance of multiple hyperplastic and dysplastic nodules that infiltrate the mammary fat pad (Fig. 2B and F), which is in accordance with the published data (22). Similar examination of whole-mount and histopathologic sections of mammary tissue derived from Cdk4(neo/neo):MMTV-neu mice showed that the ductal outgrowth and branching morphogenesis was considerably reduced compared with Cdk(+/+):MMTV-neu mice with distinctive absence of any hyperplastic or dysplastic nodules that are characteristic of the latter group of mice (Fig. 2C and G). Histopathologic examination of these mammary glands also failed to show abnormal proliferative disturbances in the mammary epithelium of Cdk4(neo/neo):MMTV-neu mice (Fig. 2C and G). This does not seem to be due to lack of Neu expression, as equal levels of Neu protein was seen in both Cdk4(+/+):MMTV-neu and Cdk4(neo/neo):MMTV-neu mice (Fig. 2J). These results suggest that Cdk4 expression is essential for the appearance of MMTV-neu–induced proliferative disturbances that are seen in Cdk4(+/+):MMTV-neu mice.

In contrast to MMTV-neu mice, the mammary glands of virgin Cdk4(+/+):MMTV-wnt mice showed precocious lobuloalvelar development that resembles that of Cdk4(+/+) pregnant female mice (Fig. 2D and H), similar to previously reported observations (23). Histopathologic examination of these mammary glands revealed extensive appearance of hyperplastic alveolar nodules, which seem to be preneoplastic lesions (23). Similar examination of whole-mount and histopathologic sections of mammary tissue derived from Cdk4(neo/neo):MMTV-wnt mice showed that the ductal outgrowth and branching morphogenesis was unaltered compared with Cdk(+/+):MMTV-wnt mice (Fig. 2E and I). Histopathologic examination of these mammary tissues again showed extensive appearance of hyperplastic alveolar nodules, similar to that seen with wild-type MMTV-wnt mice. These results indicate that MMTV-wnt–induced proliferative disturbances do not require Cdk4 expression.

Loss of expression of Cdk4 influences the incidence of mammary carcinomas. It has been previously reported that MMTV-neu–induced breast carcinomas require the expression of cyclin D1, whereas those induced by MMTV-wnt do not require the expression of cyclin D1 (16). To determine whether Cdk4 plays a similar role in the development of breast carcinomas, we monitored the four groups of transgenic mice for the appearance of breast tumors. The results of this study presented in Fig. 3A show that ∼97% of the Cdk4(+/+):MMTV-neu mice develop breast cancer between 28 to 75 weeks of age. The rest of the mice were found to develop salivary gland tumors. In sharp contrast, only ∼14% of the Cdk4(neo/neo):MMTV-neu mice develop signs of breast cancer and this incidence was found to occur only after ∼60 weeks of age; when these tumors arise, they were very small in size compared with their wild-type counterparts. Calculation of P values showed a highly significant increase in tumor frequency (P = 2.3 × 10⁻⁶) for Cdk4(+/+):MMTV-neu mice as opposed to their neo/neo counterparts. These observations suggest that development of breast tumors in MMTV-neu transgenic mice requires normal expression of Cdk4.

In contrast to Cdk4(+/+)-MMTV-neu mice, both Cdk4(+/+):MMTV-wnt mice and Cdk4(neo/neo):MMTV-wnt mice exhibited a rapid onset of breast tumors around 10 weeks of age and >90% of these mice developed breast tumors by the age of 30 weeks (Fig. 3B). Our studies also show that there was a slight delay in the development of breast tumors in Cdk4(neo/neo):MMTV-wnt mice compared with their wild-type counterparts. In contrast to the results observed for MMTV-neu mice, no significant difference in tumor frequency (P = 0.7264) was observed between Cdk4(+/+):MMTV-wnt-1 and their neo/neo counterparts. The incidence of breast tumors was seen in both male and female mice as has been previously described (22). These observations show that Cdk4 expression is dispensable for MMTV-wnt–induced breast tumorigenesis.

The histopathologic sections of the tumors is given in Fig. 3C. These sections show that Cdk4(+/+):MMTV-neu tumors have a high density of epithelial cells, whereas the tumor sections of Cdk4(neo/neo):MMTV-neu mice show increased infiltration by connective tissue. MMTV-wnt tumors in a Cdk4(+/+) or Cdk4(neo/neo) background showed a similar phenotype with a high density of epithelial cells. Interestingly, these tumors show increased vasculature, suggesting that the Wnt pathway promotes angiogenesis, which might explain the very rapid growth of tumors in these mice.

Expression patterns of Cdk4, Cdk6, and Cdk2. It has been previously shown that loss of cyclin D1 results in a breast tumor phenotype similar to that described here for Cdk4 (neo/neo) mice. In the case of MMTV-wnt:cyclin D−/− mice, loss of cyclin D1 seemed to be compensated by the overexpression of cyclin D2, which could drive the cell cycle progression (16). To understand the molecular basis for the development of breast tumors in MMTV-wnt mice in a Cdk4 null background, we examined the expression patterns and kinase activities of Cdk6 and Cdk2 in all four of the genotypes studied here. The results presented in Fig. 4A show that in both Cdk4(neo/neo):MMTV-wnt and in Cdk4(neo/neo):MMTV-neu mice, the levels of Cdk4 were undetectable, whereas the levels of Cdk4 were pronounced in Cdk4(+/+):MMTV-neu and Cdk4(+/+):MMTV-wnt mice. In contrast, the levels of Cdk6 and Cdk2 were approximately equal in all four genotypes. These results suggest that neither Cdk6 nor Cdk2 compensate for the loss of Cdk4 in MMTV-wnt transgenic mice on a Cdk4(neo/neo) background. We next examined the expression levels and the phosphorylation status of Rb in Cdk4(neo/neo), Cdk4(+/+) as well as the MMTV-neu and MMTV-wnt transgenic mice crossed to the two Cdk4 backgrounds. Results of these experiments presented in Fig. 4B show that the level of pRb Ser⁷⁸⁰ phosphorylation was low in Cdk(+/+) tissues but showed considerable elevation in those derived from both Cdk4(+/+):MMTV-neu and Cdk4(+/+):MMTV-wnt mice, which corresponds to the high levels of Cdk4, Cdk6, and Cdk2 activities seen in these tissues.

Our studies reported in this article suggest the importance of Cdk4 in mammary gland development and tumorigenesis. Whole-mount analysis and histologic sections of Cdk4(neo/neo) and Cdk4(+/+) mice show that Cdk4 is required for proper ductal branching and lobuloalveolar development of virgin female mice. In contrast, the mammary glands of cyclin D1−/− virgin females have been reported to be identical to that of wild-type mice. This difference is likely due to the compensation by cyclins D2 and D3, which are slightly up-regulated in the mammary gland of cyclin D1−/− virgin females (16). Our results suggest that in the absence of Cdk4, there is no parallel compensation by other Cdks (Fig. 4, see below). Regardless of the lack of defects in the mammary gland of cyclin D1−/− virgin females, it has been shown that cyclin D1−/− mice fail to undergo full lobuloalveolar development during late stages of pregnancy (14, 15). It has also been shown that the cyclin D1 null mice are prone to transformation induced by the wnt-1 and myc oncogenes, but not to transformation induced by the neu and ras oncogenes (16). Recent studies on MMTV-erbB2-MMTV-p16 double-transgenic mice showed that erb2-mediated tumorigenesis is blocked by p16 and that these double-transgenic mice develop rare tumors after a long delay (24). Because Cdk4(neo/neo):MMTV-neu mice showed decreased levels of ductal branching and lobuloalveolar development of the mammary glands when compared with that of Cdk4(+/+):MMTV-neu transgenic mice, we presume that Cdk4 is required for Neu-induced proliferative events that lead to ductal branching, lobuloalveolar development, and the development of hyperneoplastic alveolar nodules, and ultimately for the development of mammary tumors. We cannot rule out, however, that the observed defects in Cdk4(neo/neo) mammary gland development be the indirect result of hormonal signaling deficiencies as opposed to an epithelial cell autonomous defect.

The mammary glands of Wnt-1 transgenic virgin mice undergo precocious lobuloalveolar development and resemble the mammary glands of wild-type nontransgenic pregnant females. Our whole-mount and histologic studies of the mammary glands of Cdk4(neo/neo):MMTV-wnt-1 and Cdk4(+/+):MMTV-wnt-1 mice showed comparable lobuloalveolar development. This suggests that Cdk4 is not required for Wnt-1–induced ductal branching and lobuloalveolar development. The tumorigenesis studies conducted by us also show that both strains of mice are equally susceptible to Wnt-1-induced tumorigenesis, suggesting that Cdk4 is not required for this process. Thus, if the defect in mammary development observed in Cdk4 neo/neo females is not cell autonomous, then Wnt not only bypasses Cdk4 function, but also any conceivable defects in hormone signaling resulting from Cdk4 ablation. Our data also showed that the level of pRb phosphorylation on Ser⁷⁸⁰ correlated with G₁ Cdk activities. We have also seen that phosphorylation of this site on pRb is highly increased in breast tumor tissues independently of Cdk4 phosphorylation status, suggesting that in highly proliferative tumors, this site could be phosphorylated by Cdks other that Cdk4 (data not shown).

Considering previous results indicating that Neu may act by inducing cyclin D1 expression and our results shown here that Cdk4 is required for neu-induced tumorigenesis, we propose that the cyclin D1/Cdk4 complex is required for neu-induced tumorigenesis. It has also been suggested that wnt- and c-myc-induced breast tumorigenesis communicate with the cell cycle machinery in breast epithelial cells through different targets. In this regard, cyclin D2 expression was found to be up-regulated in tumors induced by wnt-1 and c-myc, but not neu or ras (16). Considering our data showing that Cdk4 is also dispensable for Wnt-induced tumorigenesis, and the lack of obvious compensation by other G₁ Cdks, it is tempting to speculate that Wnt signals downstream of D-type cyclin/Cdk4 complexes. In summary, our data suggest that, at least in the case of Neu-induced tumorigenesis, a Cdk4 function is required. This requirement could be for Cdk4 kinase activity, or, alternatively, for the ability of the cyclin D/Cdk4 complex to sequester p27. Further studies are necessary to differentiate between these two possibilities.

These results also have important implications with respect to therapeutic modalities that might be effective in the treatment of breast cancers that are neu-positive. The importance of Cdk4 and cyclin D1 complex in the genesis of Neu-induced breast tumors suggests that small molecule inhibitors of Cdk4 kinase activity could be very effective in blocking the growth of these human breast tumors, which often represent the most aggressive forms of human breast cancer.

Grant support: NIH P01 CA95569 and R01 AG22022.

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