Richard G. Pestell

Stat3 as an Oncogene

STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.

NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1

ABSTRACT Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of cell growth, but the molecular mechanism(s) involved in this process remains largely unknown. Here we use both a skeletal muscle differentiation model and normal diploid fibroblasts to gain insight into how NF-κB regulates cell growth and differentiation. Results obtained with the C2C12 myoblast cell line demonstrate that NF-κB functions as an inhibitor of myogenic differentiation. Myoblasts generated to lack NF-κB activity displayed defects in cellular proliferation and cell cycle exit upon differentiation. An analysis of cell cycle markers revealed that NF-κB activates cyclin D1 expression, and the results showed that this regulatory pathway is one mechanism by which NF-κB inhibits myogenesis. NF-κB regulation of cyclin D1 occurs at the transcriptional level and is mediated by direct binding of NF-κB to multiple sites in the cyclin D1 promoter. Using diploid fibroblasts, we demonstrate that NF-κB is required to induce cyclin D1 expression and pRb hyperphosphorylation and promote G1-to-S progression. Consistent with results obtained with the C2C12 differentiation model, we show that NF-κB also promotes cell growth in embryonic fibroblasts, correlating with its regulation of cyclin D1. These data therefore identify cyclin D1 as an important transcriptional target of NF-κB and reveal a mechanism to explain how NF-κB is involved in the early phases of the cell cycle to regulate cell growth and differentiation.

Transforming p21ras Mutants and c-Ets-2 Activate the Cyclin D1 Promoter through Distinguishable Regions

Several different oncogenes and growth factors promote G1 phase progression. Cyclin D1, the regulatory subunit of several cyclin-dependent kinases, is required for, and capable of shortening, the G1 phase of the cell cycle. The present study demonstrates that transforming mutants of p21ras (Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter in human trophoblasts (JEG-3), mink lung epithelial (Mv1.Lu), and in Chinese hamster ovary fibroblast cell lines. Site-directed mutagenesis of AP-1-like sequences at −954 abolished p21ras-dependent activation of cyclin D1 expression. The AP-1-like sequences were also required for activation of the cyclin D1 promoter by c-Jun. In electrophoretic mobility shift assays using nuclear extracts from cultured cells and primary tissues, several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bound the cyclin D1 −954 region. Cyclin D1 promoter activity was stimulated by overexpression of mitogen-activated protein kinase (p41MAPK) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK (p41MAPKi) or dominant negatives of ETS activation (Ets-LacZ), antagonized MAPK-dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, was antagonized by either p41MAPKi or Ets-LacZ, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21rasprovides evidence for cross-talk between the p21ras and cell cycle regulatory pathways.

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade

Caveolin‐1 is a principal component of caveolae membranes in vivo. Caveolin‐1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin‐1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin‐1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin‐1 but contain normal amounts of caveolin‐2. NIH 3T3 cells harboring antisense caveolin‐1 exhibit anchorage‐independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin‐1 downregulation is reversed when caveolin‐1 protein levels are restored to normal by loss of the caveolin‐1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin‐1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin‐1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.

Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.

Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.

The mammary gland iodide transporter is expressed during lactation and in breast cancer

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation.

ABSTRACT The RASSF1A locus at 3p21.3 is epigenetically inactivated at high frequency in a variety of solid tumors. Expression of RASSF1A is sufficient to revert the tumorigenicity of human cancer cell lines. We show here that RASSF1A can induce cell cycle arrest by engaging the Rb family cell cycle checkpoint. RASSF1A inhibits accumulation of native cyclin D1, and the RASSF1A-induced cell cycle arrest can be relieved by ectopic expression of cyclin D1 or of other downstream activators of the G1/S-phase transition (cyclin A and E7). Regulation of cyclin D1 is responsive to native RASSF1A activity, because RNA interference-mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occurs posttranscriptionally and is likely at the level of translational control. Rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G1/S-phase cell cycle progression.

p300 and p300/cAMP-response Element-binding Protein-associated Factor Acetylate the Androgen Receptor at Sites Governing Hormone-dependent Transactivation

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [14C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.

Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine‐dependent growth of hematopoietic cells

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin‐3 (IL‐3)‐dependent cell line F‐36P‐mpl, the induced expression of dominant‐negative (dn)‐STAT5 and of dn‐ras led to inhibition of IL‐3‐dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6‐STAT5A) and ras (H‐rasG12V) enabled F‐36P‐mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6‐STAT5A and H‐rasG12V individually and cooperatively transactivated the cyclin D1 promoter in luciferase assays. Both dn‐STAT5 and dn‐ras suppressed IL‐3‐induced cyclin D1 promoter activities in F‐36P‐mpl cells. Using a series of mutant cyclin D1 promoters, 1*6‐STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT‐binding sequence at −481 bp. In electrophoretic mobility shift assays, STAT5 bound to the element in response to IL‐3. Furthermore, the inhibitory effect of dn‐STAT5 on IL‐3‐dependent growth was restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine‐dependent growth of hematopoietic cells.