Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure.

A hallmark of cancer is the deregulation of cell-cycle machinery, ultimately facilitating aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 has a crucial role in the development of disease. Recently, a highly specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast cancer. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated on a panel of breast cancer cell lines. These analyses showed that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, ultimately, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies showed a highly specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F, and the ability to attenuate CDK2 activity. Acquired resistance to PD-03322991 was specifically associated with attenuation of CDK2 inhibitors, indicating that redundancy in CDK functions represents a determinant of therapeutic failure. Despite these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell-cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic.

Epidermal growth factor regulation of female-dependent CYP2A1 and CYP2C12 in primary rat hepatocyte culture.

In the present study, we describe the effects of medium composition in primary cultures of rat hepatocytes on the expression of two major constituent female-dependent CYP isoforms, CYP2C12 and CYP2A1. When female rat hepatocytes were cultured with the serum-free medium HepatoZYME, currently used to attain long-term maintenance of hepatocyte phenotypic expression, CYP2C12 mRNA and protein levels were markedly suppressed, despite the constant presence of growth hormone, the essential regulator of liver CYP2C12. Conversely, rat hepatocytes cultured in the serum-free medium Dulbecco's modified Eagle's medium-F12K, also supplemented with growth hormone, sustained near normal expression levels of CYP2C12 mRNA and protein for the 7 days of observations. Although media composition had no significant effect on mRNA expression of CYP2A1, protein content decreased dramatically in hepatocytes cultured with HepatoZYME medium. We were able to demonstrate the plasticity of the cells by restoring/suppressing the expression of CYP2C12 and CYP2A1 mRNA by reverting the culture conditions. Addition of the mitogen epidermal growth factor present in the HepatoZYME formulation to the Dulbecco's modified Eagle's medium-F12K culture media appreciably decreased expression of both CYP2C12 and CYP2A1 in female hepatocytes, while briefly sustaining levels of the cyclin inhibitor p21. Lastly, reduced CYP protein content observed in hepatocytes cultured with epidermal growth factor was not the result of an absence or reduction in the CCAAT/enhancer-binding protein alpha, a requisite transcription factor for CYP2C12 expression.