A mechanism by which calcium-induced signals are transduced to the nucleus to activate transcription of the c-fos proto-oncogene has been characterized. The serum response element (SRE), a region of the c-fos gene which controls growth factor-induced transcription, is now shown to mediate c-fos transcription in response to activation of L-type voltage-sensitive calcium channels. Calcium-dependent transcriptional activation through the SRE is mediated by the serum response factor (SRF). Membrane depolarization induces phosphorylation of SRF at Ser-103, an event shown to enhance the ability of SRF to bind the SRE. Ca(2+)-induced SRF phosphorylation occurs via a pathway that may involve Ca2+/calmodulin-dependent kinases.
The mechanisms by which Myc overexpression or Pten loss promotes prostate cancer development are poorly understood. We identified the chromatin remodeling protein, ING4, as a crucial switch downstream of Myc and Pten that is required for human prostate epithelial differentiation. Myc-induced transient expression of ING4 is required for the differentiation of basal epithelial cells into luminal cells, while sustained ING4 expression induces apoptosis. ING4 expression is lost in >60% of human primary prostate tumors. ING4 or Pten loss prevents epithelial cell differentiation, which was necessary for tumorigenesis. Pten loss prevents differentiation by blocking ING4 expression, which is rescued by ING4 re-expression. Pten or ING4 loss generates tumor cells that co-express basal and luminal markers, indicating prostate oncogenesis occurs through disruption of an intermediate step in the prostate epithelial differentiation program. Thus, we identified a new epithelial cell differentiation switch involving Myc, Pten, and ING4, which when disrupted leads to prostate tumorigenesis. Myc overexpression and Pten loss are common genetic abnormalities in prostate cancer, whereas loss of the tumor suppressor ING4 has not been reported. This is the first demonstration that transient ING4 expression is absolutely required for epithelial differentiation, its expression is dependent on Myc and Pten, and it is lost in the majority of human prostate cancers. This is the first demonstration that loss of ING4, either directly or indirectly through loss of Pten, promotes Myc-driven oncogenesis by deregulating differentiation. The clinical implication is that Pten/ING4 negative and ING4-only negative tumors may reflect two distinct subtypes of prostate cancer.
Recent studies indicate that androgen receptor (AR) signaling is critical for prostate cancer cell survival, even in castration-resistant disease wherein AR continues to function independently of exogenous androgens. Integrin-mediated adhesion to the extracellular matrix is also important for prostate cell survival. AR-positive prostate cancer cells express primarily integrin α6β1 and adhere to a laminin-rich matrix. In this study, we show that active nuclear-localized AR protects prostate cancer cells from death induced by phosphoinositide 3-kinase (PI3K) inhibition when cells adhere to laminin. Resistance to PI3K inhibition is mediated directly by an AR-dependent increase in integrin α6β1 mRNA transcription and protein expression. Subsequent signaling by integrin α6β1 in AR-expressing cells increased NF-κB activation and Bcl-xL expression. Blocking AR, integrin α6, NF-κB, or Bcl-xL concurrent with inhibition of PI3K was sufficient and necessary to trigger death of laminin-adherent AR-expressing cells. Taken together, these results define a novel integrin-dependent survival pathway in prostate cancer cells that is regulated by AR, independent of and parallel to the PI3K pathway. Our findings suggest that combined targeting of both the AR/α6β1 and PI3K pathways may effectively trigger prostate cancer cell death, enhancing the potential therapeutic value of PI3K inhibitors being evaluated in this setting.
A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.
The aims of this study were to generate a human Fab fragment against EGFR; conjugate it to paclitaxel (Taxol) as an immuno-chemotherapy agent; and investigate its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells. A431 cells (EGFR-positive), NIH 3T3 cells (EGFR-negative), and purified EGFR were used for subtractive panning on a human naïve Fab phage library to generate a human anti-EGFR Fab fragment that binds the EGFR extracellular domain in native conformation and subsequently internalizes it into the cytosol. The Fab was then conjugated with the chemotherapeutic Taxol, and cell proliferation inhibition and apoptosis (TUNEL) assays were conducted to determine the effect of this Fab-drug conjugate on A431 cells. The specificity and internalization property of this Fab were characterized by immunoprecipitation, fluorescence staining, flow cytometry, and Hum-Zap assay. The binding affinity to purified EGFR was 30 nM. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of cells underwent apoptosis after treatment with Fab-Taxol at 26 pM for 48 hours. Our findings suggest that this Fab-Taxol conjugate could be a potential immuno-chemotherapeutic drug for clinical treatment of EGFR-overexpressing tumors.
