Robert J. Trumbly

Cloning and characterization of the CYC8 gene mediating glucose repression in yeast

Mutations in the CYC8 ( = SSN6) gene of Saccharomyces cerevisiae alleviate glucose repression of many glucose-repressible genes. The gene was isolated by screening for complementation of a cyc8 effect on colony morphology. Subclones containing a 5.3-kb SalI-XbaI fragment provided complete complementation. The gene was further localized to 3.5 kb by mapping of the CYC8 mRNA and insertional mutagenesis. Insertion and deletion mutations are viable and produce the same array of phenotypes as point mutations. CYC8 disruptions also had effects on the mating ability and morphology of MAT alpha cells similar to that of tup1 mutations. The nucleotide sequence of a 4866-bp fragment, including CYC8, was determined. One long open reading frame of 966 amino acid predicts a protein of molecular weight 10,7215. The predicted protein is extremely glutamine-rich, with blocks of 16 and 31 glutamines in tandem at the N and C regions, respectively. The CYC8 gene product lacks consensus sequences for DNA-binding domains, suggesting that its function may be different from classical repressor proteins.

MUTATIONS IN LIS1 (ERG6) GENE CONFER INCREASED SODIUM AND LITHIUM UPTAKE IN SACCHAROMYCES CEREVISIAE

A Saccharomyces cerevisiae mutant, lis1-1, hypersensitive to Li+ and Na+ was isolated from a wild-type strain after ethylmethane sulfonate mutagenesis. The rates of Li+ and Na+ uptake of the mutant are about 3-4-times higher than that of the wild-type; while the rates of cation efflux from the mutant and wild-type strains are indistinguishable. The LIS1 was isolated from a yeast genomic library by complementation of the cation hypersensitivity of the lis1-1 strain. LIS1 is a single copy, nonessential gene. However, the deletion of LIS1 from the wild-type results in a growth defect in addition to the cation hypersensitive phenotype. The order of increasing cation uptake rates of the wild-type and mutant strains, LIS1 < lis1-1 < lis1-delta 1::LEU2, correlates perfectly with the degree of cation hypersensitivity, suggesting that the cation hypersensitivity is primarily due to increased rates of cation influx. LIS1 encodes a membrane associated protein 384 amino acids long. Data base searches indicate that LIS1 is identical to ERG6 in S. cerevisiae which encodes a putative S-adenosylmethionine-dependent methyltransferase in the ergosterol biosynthetic pathway. Cell membranes of lis1 (erg6) mutants are known to be devoid of ergosterol and have altered sterol composition. Since membrane sterols can influence the activity of cation transporters, the increased cation uptake of the lis1 mutants may stem from an altered function of one or many different membrane transporters.

RKIP inhibits NF-κB in cancer cells by regulating upstream signaling components of the IκB kinase complex

MINT‐7386121: TRAF6 (uniprotkb:Q9Y4K3) physically interacts (MI:0915) with RKIP (uniprotkb:P30086) by anti bait co‐immunoprecipitation (MI:0006)

Multiple regulatory proteins mediate repression and activation by interaction with the yeast Mig1 binding site

A major mediator of glucose repression in yeast is Mig1, a zinc finger protein that binds to a GC‐rich recognition sequence found upstream of many glucose‐repressible genes. Because these Mig1 sites are found upstream of genes under different modes of regulation, we studied regulation of transcription mediated by an isolated Mig1 site placed upstream of a reporter gene under control of UASCYC1. The Mig1 site responded appropriately to glucose control and regulatory mutations, including snf1, reg1, cyc8, and tup1, mimicking the behavior of the SUC2 gene. Deletion of the MIG1‐coding gene reduced but did not eliminate glucose repression mediated by the Mig1 site. Complete loss of repression was seen in a mig1 mig2 double mutant. When the UASCYC1 was replaced by UASADH1 in the reporter plasmid, the Mig1 site activated transcription under most conditions. Mutations of the two Mig1 binding sites in the SUC2 promoter resulted in loss of activation of SUC2 expression. These results suggest the presence of an unknown activator or activators that binds to the Mig1 site. The activator is not any of the proteins previously proposed to bind to this site, including Mig1, Mig2, Msn2, or Msn4. Band shift assays showed that Mig1 is the major protein in yeast cell extracts that binds to the Mig1 site in vitro. This binding is not regulated by glucose or mutations in CYC8 or TUP1.

The ETS Domain Transcription Factor Elk1 Directs a Critical Component of Growth Signaling by the Androgen Receptor in Prostate Cancer Cells

Background: Mechanisms that redirect androgen receptor signaling to primarily support prostate tumor growth are poorly understood. Results: Prostate cancer cells were addicted to ELK1, which tethered AR to activate growth genes in hormone-dependent and castration-recurrent PC without ELK1 phosphorylation. Conclusion: ELK1 directs a critical arm of transcriptional growth signaling by AR that is preserved in CRPC. Significance: The ELK1-AR interaction offers a functionally tumor-selective drug target. The androgen receptor (AR) is essential for diverse aspects of prostate development and function. Molecular mechanisms by which prostate cancer (PC) cells redirect AR signaling to genes that primarily support growth are unclear. A systematic search for critical AR-tethering proteins led to ELK1, an ETS transcription factor of the ternary complex factor subfamily. Although genetically redundant, ELK1 was obligatory for AR-dependent growth and clonogenic survival in both hormone-dependent PC and castration-recurrent PC cells but not for AR-negative cell growth. AR required ELK1 to up-regulate a major subset of its target genes that was strongly and primarily enriched for cell growth functions. AR functioned as a coactivator of ELK1 by association through its A/B domain, bypassing the classical mechanism of ELK1 activation by phosphorylation and without inducing ternary complex target genes. The ELK1-AR synergy per se was ligand-independent, although it required ligand for nuclear localization of AR as targeting the AR A/B domain to the nucleus recapitulated the action of hormone; accordingly, Casodex was a poor antagonist of the synergy. ELK3, the closest substitute for ELK1 in structure/function and genome recognition, did not interact with AR. ELK1 thus directs selective and sustained gene induction that is a substantial and critical component of growth signaling by AR in PC cells. The ELK1-AR interaction offers a functionally tumor-selective drug target.

On the regulation of Na+/H+ and K+/H+ antiport in yeast mitochondria: Evidence for the absence of an Na+-selective Na+/H+ antiporter

Unlike mammalian mitochondria, yeast mitochondria swell spontaneously in both NaOAc and KOAc. This swelling reflects the activity of an electroneutral cation/H+ antiport pathway. Transport of neither salt is stimulated by depletion of endogenous divalent cations; however, it can be inhibited by addition of exogenous divalent cations (Mg2+ IC50 = 2.08 mM, Ca2+ IC50 = 0.82 mM). Transport of both Na+ and K+ can be completely inhibited by the amphiphilic amines propranolol (IC50 = 71 microM) and quinine (IC50 = 199 microM) with indistinguishable IC50 values. Dicyclohexylcarbodiimide inhibits with a second-order rate constant of 1.6 x 10(-4) (nmol DCCD/mg)-1 min-1 at 0 degrees C; however, with both Na+ and K+ inhibition reaches a maximum of about 46%. The remaining transport can still be inhibited by propranolol. Transport of both cations is sensitive to pH; yielding linear Hill plots and Dixon plots with a pIC50 value of 7.7 for both Na+ and K+. These properties are qualitatively the same as those of the non-selective K+/H+ antiporter of mammalian mitochondria. However, the remarkable similarity between the data obtained in Na+ and K+ media suggests that an antiporter akin to the Na(+)-selective Na+/H+ antiporter of mammalian mitochondria, which is inhibited by none of these agents, is absent in yeast. In an attempt to reveal the activity of a propranolol-insensitive Na(+)-selective antiporter, we compared the rates of Na+/H+ and K+/H+ antiport in the presence of sufficient propranolol to block the K+/H+ antiporter. Between pH 4.6 and 8.8 no difference could be detected. Consequently, we conclude that yeast mitochondria lack the typical Na(+)-selective Na+/H+ antiporter of mammalian mitochondria.

C/EBPα redirects androgen receptor signaling through a unique bimodal interaction

Nuclear expression of CCAAT enhancer binding protein-α (C/EBPα), which supports tissue differentiation through several antiproliferative protein–protein interactions, augurs terminal differentiation of prostate epithelial cells. C/EBPα is also a tumor suppressor, but in many tumors its antiproliferative interactions may be attenuated by de-phosphorylation. C/EBPα acts as a corepressor of the classical androgen response element (ARE)-mediated gene activation by the androgen receptor (AR), but this is paradoxical as the genotropic actions of AR are crucial not only for the growth of the prostate but also for its maintenance and function. We show that DNA-bound C/EPBα recruits AR to activate transcription. C/EBPα-dependent trans-activation by AR also overrode suppression of AREs by C/EBPα elsewhere in a promoter. This mechanism was remarkable in that its androgen dependence was apparently for nuclear translocation of AR; it was otherwise androgen independent, flutamide insensitive and tolerant to disruption of AR dimerization. Gene response profiles and global chromatin associations in situ supported the direct bimodal regulation of AR transcriptional signaling by C/EBPα. This unique mechanism explains the functional coordination between AR and C/EPBα in the prostate and also shows that hormone-refractory AR signaling in prostate cancer could occur through receptor tethering.

Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements

A need for androgen response elements (AREs) for androgen receptor (AR)-dependent growth of hormone depletion-insensitive prostate cancer is generally presumed. In such cells, androgen-independent activation by AR of certain genes has been attributed to selective increases in basal associations of AR with putative enhancers. We examined the importance of AR binding to DNA in prostate cancer cells in which proliferation in the absence of hormone was profoundly (∼ 90%) dependent on endogenous AR and where the receptor was not up-regulated or mutated but was predominantly nuclear. Here, ARE-mediated promoter activation and the binding of AR to a known ARE in the chromatin remained entirely androgen dependent, and the cells showed an androgen-responsive gene expression profile with an unaltered sensitivity to androgen dose. In the same cells, a different set of genes primarily enriched for cell division functions was activated by AR independently of hormone and significantly overlapped the signature gene overexpression profile of hormone ablation-insensitive clinical tumors. After knockdown of endogenous AR, hormone depletion-insensitive cell proliferation and AR apoprotein-dependent gene expression were rescued by an AR mutant that was unable to bind to ARE but that could transactivate through a well-established AR tethering protein. Hormone depletion-insensitive AR binding sites in the chromatin were functional, binding, and responding to both the wild-type and the mutant AR and lacked enrichment for canonical or noncanonical ARE half-sites. Therefore, a potentially diverse set of ARE-independent mechanisms of AR interactions with target genes must underlie truly hormone depletion-insensitive gene regulation and proliferation in prostate cancer.

During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein

IntroductionCurrent hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear.MethodsIn estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR.ResultsBasal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-β and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions.ConclusionsIn hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.

RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of-function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.