These data suggest that combined treatment with drugs that target EGFR and ErbB-2 might result in an efficient inhibition of tumor growth in those breast carcinoma patients whose tumors co-express both receptors.

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.

Resveratrol (3,4',5-trans-trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol can induce growth inhibition and apoptosis in MDA-MB-231, a highly invasive and metastatic breast cancer cell line, in concomitance with a dramatic endogenous increase of growth inhibitory/proapoptotic ceramide. We found that accumulation of ceramide derives from both de novo ceramide synthesis and sphingomyelin hydrolysis. More specifically we demonstrated that ceramide accumulation induced by resveratrol can be traced to the activation of serine palmitoyltransferase (SPT), the key enzyme of de novo ceramide biosynthetic pathway, and neutral sphingomyelinase (nSMase), a main enzyme involved in the sphingomyelin/ceramide pathway. However, by using specific inhibitors of SPT, myriocin and L-cycloserine, and nSMase, gluthatione and manumycin, we found that only the SPT inhibitors could counteract the biological effects induced by resveratrol. Thus, resveratrol seems to exert its growth inhibitory/apoptotic effect on the metastatic breast cancer cell line MDA-MB-231 by activating the de novo ceramide synthesis pathway.

Resistance to the growth-inhibitory action of retinoic acid (RA), the bioactive derivative of vitamin A, is common in human tumors. One form of RA resistance has been associated with silencing and hypermethylation of the retinoic acid receptor ␤2 gene (RAR␤2), an RA-regulated tumor suppressor gene. The presence of an epigenetically silent RAR␤2 correlates with lack of the RA receptor ␣ (RAR␣). Normally, RAR␣ regulates RAR␤2 transcription by mediating dynamic changes of RAR␤2 chromatin in the presence and absence of RA. Here we show that interfering with RA signal through RAR␣ (which was achieved by use of a dominantnegative RAR␣, by downregulation of RAR␣ by RNA interference, and by use of RAR␣ antagonists) induces an exacerbation of the repressed chromatin status of RAR␤2 and leads to RAR␤2 transcriptional silencing. Further, we demonstrate that RAR␤2 silencing causes resistance to the growth-inhibitory effect of RA. Apparently, RAR␤2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate that restoration of RA signal at a silent RAR␤2 through RAR␣ leads to RAR␤2 reactivation. This report provides proof of principle that RAR␤2 silencing and RA resistance are consequent to an impaired integration of RA signal at RAR␤2 chromatin.