Lora W. Forman

Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells☆

The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of < or =30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold+/-1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.

Protein Kinase Cδ Is Required for Survival of Cells Expressing Activated p21RAS

Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21RAS results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21RAS oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21RAS. Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCδ isozyme demonstrated that PKCδ plays a critical role in p21RAS-mediated apoptosis. An activating p21RAS mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKCδ protein and activity in cells, whereas inhibition of p21RAS activity decreased the expression of the PKCδ protein. Activation of the Akt survival pathway by oncogenic Ras required PKCδ activity. Akt activity was dramatically decreased after PKCδ suppression in cells containing activated p21RAS. Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKCδ inhibition. Collectively, these findings demonstrate that p21RAS, through its downstream effector PI3K, induces PKCδ expression and that this increase in PKCδ activity, acting through Akt, is required for cell survival. The p21RAS effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCδ activity was also determined to be PI3K. PI3K (p110CAAX, where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKCδ inhibition. Thus, the same p21RAS effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKCδ and Akt. Selective suppression of PKCδ activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21RAS.

Prohibitin and the SWI/SNF ATPase subunit BRG1 are required for effective androgen antagonist-mediated transcriptional repression of androgen receptor-regulated genes

Androgen antagonists or androgen deprivation are the primary therapeutic modalities for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this study, we identify two molecules which are required for effective prostate cancer cell responsiveness to androgen antagonists. We establish that androgen receptor (AR)-dependent transcriptional suppression by androgen antagonists requires the tumor suppressor prohibitin. This requirement for prohibitin was demonstrated using structurally-distinct androgen antagonists, stable and transient knockdown of prohibitin and transfected and endogenous AR-responsive genes. The SWI-SNF complex core ATPase BRG1, but not its closely-related counterpart ATPase BRM, is required for this repressive action of prohibitin on AR-responsive promoters. Androgen antagonists induce recruitment of prohibitin and BRG1 to endogenous AR-responsive promoters and induce a physical association between AR and prohibitin and BRG1. The recruitment of prohibitin to endogenous AR-responsive promoters is dependent upon antagonist-bound AR. Prohibitin binding in the prostate-specific antigen (PSA) promoter results in the recruitment of BRG1 and the dissociation of p300 from the PSA promoter. These findings suggest that prohibitin may function through BRG1-mediated local chromatin remodeling activity and the removal of p300-mediated acetylation to produce androgen antagonist-mediated transcriptional repression. Furthermore, in addition to its necessary role in AR-mediated transcriptional repression, we demonstrate that prohibitin is required for full and efficient androgen antagonist-mediated growth suppression of prostate cancer cells.

Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras.

The induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations.

Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

BackgroundA subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.MethodsshRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA.ResultsWe demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.ConclusionsThese findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki -Ras

The product of the ras proto-oncogene has been implicated as an essential signal transducer, involved in a variety of biological or pathological activities, including apoptosis. The aim of this investigation was to further explore the mechanisms of apoptosis triggered by Ras. Stable expression of constitutively-activated (v-)Ki-Ras in Balb/c-3T3 mouse fibroblasts resulted in a loss of G1 arrest in response to treatments which induced cell cycle arrest in the parental Balb/c-3T3 cells, accompanied by decreased expression of the p53 tumor suppressor protein and the GADD45 gene, the product of which is involved in DNA repair, and deregulated expression of the MDM-2 gene, the product of which can regulate p53 expression. Ki-Ras expression also increased the frequency of PALA-selectable CAD gene amplification, and paradoxically the susceptibility to PALA-induced apoptosis. After persistent serum-starvation, cells expressing the activated ras gene lost clonogenic potential, indicating impaired capability for genetic repair in the cells. Taken together, these data suggest that activated Ki-ras may confer genetic instability upon cells, possibly through interference with tumor suppressors, such as p53. While this instability may facilitate adaptation to environmental stresses, this instability in the genome also renders cells containing activated ras genes intrinsically more susceptible to programmed cell death, possibly by accumulation of undesirable or lethal genetic events during the process of tumor development.

Identification of LTR‐specific small non‐coding RNA in FeLV infected cells

The U3‐LTR region of leukemia viruses transactivates cancer‐related signaling pathways through the production of a non‐coding RNA transcript although the role of this transcript in virus infection remains unknown. In this study we demonstrate for the first time that an long terminal repeat (LTR)‐specific small non‐coding RNA is produced from a feline leukemia virus (FeLV)‐infected feline cell line. RNA cloning identified this as a 104 base transcript that originates from the U3‐LTR region. We also demonstrate that in in vitro assays this LTR‐RNA transcript activates NFκB signaling. Taken together, our findings suggest a possible role for this LTR transcript in FeLV pathogenesis.

Protein kinase Cδ inactivation inhibits cellular proliferation and decreases survival in human neuroendocrine tumors

The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21(Ras)-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI(3)-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21(Ras) signaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24  h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.