Mammary gland development can be divided into seven stages: embryonic, postnatal, juvenile, puberty, pregnancy, lactation, and involution. One of the leading risk factors for breast cancer is nullparity . Hence, the delineation of factors that regulate lactogenesis (terminal differentiation) is important in understanding the etiology of breast cancer.
Excess activation of signaling pathways downstream of the epidermal growth factor receptor, ErbB1, has been directly linked to breast cancer development and chemotherapeutic resistance . While EGF is required for normal mammary epithelial cell proliferation, it has been shown to inhibit lactogenic differentiation of HC11 mammary epithelial cells both in vitro and in vivo, concomitant with stimulation of the Ras/Mek/Erk and the PI-3-kinase pathways [6, 8, 9, 12]. The PI-3-kinase pathway is important in tumorigenesis in several ways. Aberrant PI-3-kinase activation has been demonstrated to promote both proliferation and survival of transformed cells, including those exhibiting EGF-dependent transformation. The mutation and deregulation of PI-3-kinase pathway components has recently been linked to a number of human malignancies [33–38] and breast cancer associated mutations of the p110 catalytic subunit of PI-3-kinase were oncogenic when tested in immortalized mammary epithelial cells . Elevated Akt levels have been found in breast, ovarian, colon and thyroid cancers [54, 55].
The data reported here confirm and extend our earlier results indicating that PI-3-kinase inhibitors rescue the EGF-induced block of β-casein promotor-regulated luciferase activity, β-casein transcription and mammosphere formation in lactogen-treated HC11 cells. Furthermore, the expression of a conditionally active-Akt1 blocked lactogenic differentiation, whereas dominant negative-Akt1 enhanced it. These results indicate that EGF blocks HC11 lactogenic differentiation in part via a PI-3-kinase/Akt dependent mechanism. In addition, our data indicate that in HC11 cells PI-3-kinase regulated the EGF-dependent transcription of cyclin D1 and osteopontin (OPN) (Wang, Galbaugh, and Cutler, unpublished observation), both of which are regulated by the PI-3-kinase pathway in tumor cells [56, 57]. However, PI-3-kinase inhibition did not entirely prevent the EGF-induced reduction in transcription of differentiation specific target genes. For example, EGF blocks transcription of prolactin-induced protein, PIP, via the Mek/Erk and not PI-3-kinase pathways (Wang, Galbaugh and Cutler, unpublished data). Consequently, we conclude that the involvement of the PI-3-kinase pathway in blocking lactogenic differentiation is partly independent of Stat5-induced transcriptional changes.
The inhibitory effect of PI-3-kinase on β-casein transcription and β-casein promotor luciferase activity is likely through combined mechanisms involving signals mediated by prolactin and dexamethasone. Dexamethasone can play a role in inhibiting the phosphorylation of p70S6 kinase thereby decreasing protein synthesis . Our study reveals that dexamethasone inhibits the phosphorylation of p70S6 kinase in HC11 cells. This suggests a role for dexamethasone in lactogenic hormone-induced differentiation in addition to its role in activating glucocorticoid receptor, which acts synergistically with Stat5 to initiate β-casein transcription [58–60]. PI-3-kinase mediated translational control influences differentiation in erythroid precursers. Stem cell factor delays differentiation of erythroid precursers in part by activating PI-3-kinase pathway resulting in 4E-BP1 phosphorylation and the subsequent recruitment of growth-specific mRNAs into polysomes ; and ectopic expression of eIF4E in these cells has the same effect . Our work has not identified specific protein targets whose synthesis is translationally regulated by the PI-3-kinase/Akt/mTOR pathway in HC11 cells. However, a recent study demonstrated that ErbB2 increases the synthesis of the vascular endothelial growth factor (VEGF) protein via the activation of mTOR and p70S6K in human breast cancer cells . This finding suggests that it may be essential to down regulate VEGF or other growth factors in order for lactogenic differentiation to proceed. Also, SOCS-1 can be translationally repressed via a cap-dependent mechanism , suggesting that another effect of activation of PI-3-kinase pathway may be the elevation of SOCS-1 and inhibition of prolactin-induced Jak-Stat signaling.
Through the use of chemical inhibitors, alone or in combination, our data revealed that the PI-3-kinase and Mek/Erk signaling pathways are independent and synergistic in their block of HC11 lactogenic differentiation. We determined that EGF activates phosphorylation of Akt, mTOR, p70S6 kinase, ribosomal protein S6, eIF4E and 4E-BP1 in a PI-3-kinase dependent manner, and PI-3-kinase activation may prevent lactogenic differentiation in HC11 mammary epithelial cells by regulating the synthesis of proteins.
While several studies have suggested that Erk activation can be regulated through the PI-3-kinase pathway [65, 66] our data demonstrated that EGF stimulation of Erk activation in HC11 mammary epithelial cells was not altered by blocking PI-3-kinase signaling with LY294002. In addition, our previous work revealed that PI-3-kinase activation by EGF receptor proceeded without requiring Ras activation . A report by Bailey et al. demonstrated that low level activation of Akt by prolactin stimulation blocked the inhibitory effects of exogenous TGFβ on HC11 cells . Our study examined the effects of stronger Akt activation by mitogen rather than by TGFβ, which induces apoptosis in HC11 cells. While no previous studies have addressed the mechanism by which PI-3-kinase blocks lactogenic differentiation, we demonstrated that the inhibition of PI-3-K, Akt or mTOR blocked the activation of p70S6 kinase and its downstream targets. We also demonstrated that the expression of a conditionally active-Akt1 leads to the constitutive activation of p70S6 kinase. Interestingly, we discovered that PDK1 is constitutively phosphorylated in HC11 cells and this is not blocked by LY294002. While PDK1 has been shown to directly activate p70S6 kinase independently of Akt , our results indicate that the activation of p70S6 kinase is dependent on Akt and mTOR in HC11 cells.
The present study enhances our knowledge of HC11 mammary epithelial differentiation in several ways. We demonstrated that Akt activation can inhibit lactogenic hormone induced differentiation in mammary epithelial cells. Two previous studies questioned whether PI-3-kinase activation of Akt in normal mammary epithelial cells is sufficient for cellular transformation [68, 69]. Our observation that blocking the activation of PI-3-kinase restored mammosphere formation, which was inhibited by EGF, is in agreement with reports that conditionally active-Akt1 promotes large and misshapen acinar structures in MCF-10A cells [39, 40]. However, the results obtained from cell culture experiments are somewhat different from in vivo analysis of Akt. Akt is expressed during lactation in vivo at a point when levels of other kinases are diminishing . The expression presumably plays a critical function in cell survival at this point in mammary differentiation. The transgenic expression of MMTV-CA-Akt enhanced/temporally extended the expression of β-casein and resulted in more differentiated cells surviving in the tissue during lactation again at the time when other receptor tyrosine kinases were nearly absent [71, 72]. Recently Jankiewitz et al. demonstrated that treatment of lactating mice with rapamycin decreased the size of the mammary glands and inhibited HC11 differentiation by blocking lactogenic hormone-induced expression of the transcriptional regulator Id2 . Our HC11 experiments were performed in immortalized HC11 cells grown in the presence of insulin and fetal bovine serum, sources of stimulation for other receptor tyrosine kinases including those required for cell survival. We also found that blocking PI-3-kinase signaling with chemical inhibitors in the absence of additional mitogen decreased HC11 lactogenic differentiation. However, the stimulation of downstream pathways by EGF or CA-Akt was in excess of the normal cell survival signaling and thereby altered cell responses accordingly. Our results indicate that activation of p70S6 kinase under those conditions is detrimental to HC11 lactogenic differentiation. While this study presents a comprehensive investigation of the role that EGF-induced PI-3-kinase and Akt play in HC11 lactogenic differentiation, further studies in animal models will provide a greater understanding of the role of PI-3-kinase and p70S6 kinase on ErbB1 signals during hormonal regulation of the mammary gland.