Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells.

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or "track" formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm(2). Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm(2). AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.

Non-oncogene Addiction to SIRT3 Plays a Critical Role in Lymphomagenesis.

Diffuse large B cell lymphomas (DLBCLs) are genetically heterogeneous and highly proliferative neoplasms derived from germinal center (GC) B cells. Here, we show that DLBCLs are dependent on mitochondrial lysine deacetylase SIRT3 for proliferation, survival, self-renewal, and tumor growth in vivo regardless of disease subtype and genetics. SIRT3 knockout attenuated B cell lymphomagenesis in VavP-Bcl2 mice without affecting normal GC formation. Mechanistically, SIRT3 depletion impaired glutamine flux to the TCA cycle via glutamate dehydrogenase and reduction in acetyl-CoA pools, which in turn induce autophagy and cell death. We developed a mitochondrial-targeted class I sirtuin inhibitor, YC8-02, which phenocopied the effects of SIRT3 depletion and killed DLBCL cells. SIRT3 is thus a metabolic non-oncogene addiction and therapeutic target for DLBCLs.

Synergistic antiangiogenic effects of stathmin inhibition and taxol exposure.

Stathmin is one of the key regulators of the microtubule cytoskeleton and the mitotic spindle in eukaryotic cells. It is expressed at high levels in a wide variety of human cancers and may provide an attractive target for cancer therapy. We had previously shown that stathmin inhibition results in the abrogation of the malignant phenotype. The microtubule-interfering drug, taxol, has both antitumorigenic and antiangiogenic properties. We had also shown that the antitumor activities of taxol and stathmin inhibition are synergistic. We hypothesized that taxol and stathmin inhibition may also have synergistic antiangiogenic activities. A replication-deficient bicistronic adenoviral vector that coexpresses green fluorescent protein and an anti-stathmin ribozyme was used to target stathmin mRNA. Exposure of endothelial cells to anti-stathmin adenovirus alone resulted in a dose-dependent inhibition of proliferation, migration, and differentiation into capillary-like structures. This inhibition was markedly enhanced by exposure of transduced endothelial cells to very low concentrations of taxol, which resulted in a virtually complete loss of proliferation, migration, and differentiation of endothelial cells. In contrast, exposure of nontransduced endothelial cells to taxol alone resulted in a modest inhibition of proliferation, migration, and differentiation. Our detailed analysis showed that the antiangiogenic effects of the combination of stathmin inhibition and taxol exposure are synergistic. Our studies also showed that the mechanism of this synergistic interaction is likely to be mediated through the stabilization of microtubules. Thus, this novel combination may provide an attractive therapeutic strategy that combines a synergistic antitumor activity with a synergistic antiangiogenic activity.

Therapeutic interactions between stathmin inhibition and chemotherapeutic agents in prostate cancer.

Limitations of prostate cancer therapy may be overcome by combinations of chemotherapeutic agents with gene therapy directed against specific proteins critical for disease progression. Stathmin is overexpressed in many types of human cancer, including prostate cancer. Stathmin is one of the key regulators of the microtubule network and the mitotic spindle and provides an attractive therapeutic target in cancer therapy. We recently showed that adenovirus-mediated gene transfer of anti-stathmin ribozyme could suppress the malignant phenotype of prostate cancer cells in vitro. In the current studies, we asked whether the therapeutic effects of stathmin inhibition could be further enhanced by exposure to different chemotherapeutic agents. Exposure of uninfected LNCaP human prostate cancer cells or cells infected with a control adenovirus to Taxol, etoposide, 5-fluorouracil (5-FU), or Adriamycin resulted in modest decrease in proliferation and clonogenicity. Interestingly, exposure of cells infected with an anti-stathmin adenovirus to Taxol or etoposide resulted in a complete loss of proliferation and clonogenicity, whereas exposure of the same cells to 5-FU or Adriamycin potentiated the growth-inhibitory effects of the anti-stathmin ribozyme, but the cells continued to proliferate. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis of uninfected cells or cells infected with a control adenovirus showed modest induction of apoptosis in the presence of different drugs. In contrast, cells infected with the anti-stathmin adenovirus showed a marked increase in apoptosis on exposure to Taxol or etoposide and a modest increase on exposure to 5-FU or Adriamycin. Overall, the effects of combinations of anti-stathmin ribozyme with Taxol or etoposide were synergistic, whereas the effects of combinations of anti-stathmin ribozyme with 5-FU or Adriamycin were additive. Moreover, triple combination of anti-stathmin ribozyme with low noninhibitory concentrations of Taxol and etoposide resulted in a profound synergistic inhibition of proliferation, clonogenicity, and marked induction of apoptosis. This synergy might be very relevant for the treatment of prostate cancer because Taxol and etoposide are two of the most effective agents in this disease. Thus, this combination may provide a novel form of prostate cancer therapy that would avoid toxicities associated with the use of multiple chemotherapeutic agents at full therapeutic doses.

Targeting stathmin in prostate cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin is expressed at high levels in a variety of human cancers and provides an attractive molecule to target in cancer therapies that disrupt the mitotic apparatus. We developed replication-deficient bicistronic adenoviral vectors that coexpress green fluorescent protein and ribozymes that target stathmin mRNA. The therapeutic potential of these recombinant adenoviruses was tested in an experimental androgen-independent LNCaP prostate cancer model. Adenovirus-mediated transfer of anti-stathmin ribozymes resulted in efficient transduction and marked inhibition of stathmin expression in these cells. Cells that were transduced with the anti-stathmin adenoviruses showed a dramatic dose-dependent growth inhibition. This was associated with accumulation of LNCaP cells in the G2-M phases of the cell cycle. A similar dose-dependent inhibition of clonogenic potential was also observed in cells infected with anti-stathmin adenoviruses. Morphologic and biochemical analysis of infected cells showed a marked increase in apoptosis characterized by detachment of the cells, increased chromatin condensation, activation of caspase-3, and fragmentation of internucleosomal DNA. If these findings are confirmed in vivo, it may provide an effective approach for the treatment of prostate cancer.

Role of stathmin in the regulation of the mitotic spindle: potential applications in cancer therapy.

Stathmin is a member of a novel class of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin promotes microtubule depolymerization during interphase and late mitosis. This microtubule depolymerizing activity of stathmin is regulated by changes in its level of phosphorylation that occur during cell cycle progression. These modifications allow it to play a critical role in the regulation of the dynamic equilibrium of microtubules during different phases of the cell cycle. Stathmin is expressed at high levels in a wide variety of human cancers. Inhibition of stathmin expression in malignant cells interferes with their orderly progression through the cell cycle and abrogates their transformed phenotype. Thus, stathmin provides an attractive molecular target for disrupting the mitotic apparatus and arresting the growth of malignant cells. In this review, we describe the current understanding of the role of stathmin in the regulation of the mitotic spindle and discuss its potential as a therapeutic target of cancer therapy.

New paradigms in microtubule-mediated endocrine signaling in prostate cancer.

Metastatic prostate cancer has limited therapeutic options and has remained a major clinical challenge. Historically, prostate cancer has been widely recognized as a chemotherapy-resistant disease. However, clinical studies with anti-microtubule agents over the past decade have shown important efficacy in improving survival in patients with advanced disease. The favorable outcomes with microtubule-targeted agents have thus rekindled interest in such therapies for the clinical management of prostate cancer. Microtubules are dynamic polymers of tubulin molecules that play diverse roles within the cell. The dynamic property of microtubules is responsible for forming the bipolar mitotic apparatus, the mitotic spindle, that functions to precisely segregate the chromosomes during cell division. Thus, owing to the pivotal role that they play in the orchestration of mitotic events, microtubules provide excellent targets for anti-cancer therapy. Recent evidence also suggests that microtubules play a crucial role in the regulation of endocrine signaling pathways. Interestingly, microtubule-targeted agents such as taxanes not only inhibit cell division but also impair endocrine receptor signaling in prostate cancer. Herein, we provide an overview of the current status of microtubule-targeted therapies that are used in the treatment of prostate cancer and discuss novel mechanisms by which such therapies modulate endocrine signaling in prostate cancer. We also address the emerging roles of microtubule regulatory proteins in prostate carcinogenesis that could serve as attractive targets for prostate cancer therapy and might also serve as predictive biomarkers to identify patients who may benefit from endocrine and/or chemotherapy. This may have important implications in designing mechanism-based and targeted-therapeutic strategies for prostate cancer.