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Pathways associated with G9a copy number in the Leeds melanoma cohort

Primer sequences used in this study

IC50 values of G9a inhibitors (UNC0638 and BIX01294) in melanoma cell lines carrying different G9a copy number and H3K9me2 status

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations.Significance:Oncogenic G9a abnormalities drive tumorigenesis and the “cold” immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore “hot” tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890

Whole exome sequence datasets used to discover G9a recurrent mutations

GSEA for KEGG pathway genesets G9a-knockdowned Hs944T cells

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. Significance: Oncogenic G9a abnormalities drive tumorigenesis and the “cold” immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore “hot” tumor immune microenvironments. This article is highlighted in the In This Issue feature, p. 890

Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. The detrusor muscle samples showed diabetes-induced increased tissue remodeling-type events such as Actin Cytoskeleton Signaling and Signaling by Rho Family GTPases. The diabetic urothelium samples exhibited oxidative stress responses, as seen in the suppression of protein expression for key players in the NRF2-Mediated Oxidative Stress Response pathway. These results suggest that diabetes induced elevated inflammatory responses, oxidative stress, and tissue remodeling are involved in the development of tissue specific diabetic bladder dysfunctions. Validation of signaling dysregulation as a function of diabetes was performed using Western blotting. These data illustrated changes in ERK1/2 phosphorylation as a function of diabetes, with significant decreases in diabetes-associated phosphorylation in urothelium, but the opposite effect in detrusor muscle. These data highlight the importance of understanding tissue specific effects of disease process in understanding pathophysiology in complex disease and pave the way for future studies to better understand important molecular targets in reversing bladder dysfunction.

Phorbol 12-myristate 13-acetate (PMA)-induced apoptosis of androgen sensitive LNCaP human prostate cancer cells is a well known phenomenon that involves prolonged translocation of multiple protein kinase C (PKC) isozymes to nonnuclear membranes. We have shown recently that PMA-induced death of C4-2 cells, androgen hypersensitive derivatives of LNCaP cells, requires both PKCdelta and a redundant pathway that includes PKCs alpha and epsilon. In contrast, it has been reported that overexpression of murine PKCepsilon in LNCaP cells renders those cells resistant to PMA-induced death, as well as androgen insensitive. Here we report that inducible or constitutive overexpression of human PKCepsilon does not alter the sensitivity of LNCaP cells to either PMA or androgen, nor does it alter expression of caveolin-1 or phosphorylated Rb, reported effects of overexpression of murine PKCepsilon. Moreover, overexpression of very high amounts of PKCepsilon sensitized LNCaP cells to induction of apoptosis by bryostatin 1, a non tumor-promoting activator and down-regulator of PKC isozymes that blocks PMA-induced apoptosis of parental LNCaP cells, mimicked our previous results with overexpression of PKCalpha in LNCaP cells. Given reports that overexpression of PKCepsilon is frequent in human prostate tumors, our results may have important implications for a potential prostate cancer therapy.

Metastasis is a vital target for cancer treatment, since the majority of cancer patients die from metastatic, rather than the primary disease. KiSS1 has been identified as a metastasis suppressor gene in melanoma and breast carcinomas. We show here that KiSS1 is also a metastasis suppressor in human ovarian cancer. Overexpression of KiSS1 in ovarian cancer cells inhibits cell migration induced by serum or lysophosphatidic acid (LPA), and colonization in soft agar, but not cell proliferation, representing the characteristics of a metastasis suppressor gene. Furthermore, using an experimental metastatic mouse model, we show that expression of KiSS1 in SKOV3 ovarian cancer cells suppresses >50% metastatic colonization in mice (P < 0.0001). We find that activating protein kinase C (PKC) reverses about 80% of the inhibited cell migration induced by KiSS1, while down-regulation of PKCalpha with shRNA restores KiSS1 effect, providing evidence that inhibiting PKCalpha may be an important mechanism of the effect of KiSS1. These results suggest that KiSS1 is a metastasis suppressor of ovarian cancer and may be a potential molecular target for the treatment.

Protein kinase C (PKC) zeta is a phospholipid-dependent serine/threonine kinase that appears to perform important cell signaling functions. Two forms of PKC zeta RNA, with different 5' ends, have been reported. The major form (zeta) is expressed in most, if not all tissues, while the minor form (zeta'), which encodes the catalytic domain of the enzyme without most of its regulatory domain, is predominant in normal brain and certain rat prostate tumors. We report here the structure of the 5' end of the rat PKC zeta gene, demonstrating that both forms of RNA can be transcribed from the same gene through the use of alternative promoters and splicing. In luciferase reporter constructs, progressive deletions of the PKC zeta and zeta' 5' flanking sequences yielded activities that were higher in the cell lines expressing endogenous PKC zeta and zeta' RNAs, respectively. Also, multiple PCRs across different introns of the PKC zeta gene indicate that recent duplication of the gene or the existence of a closely related pseudogene in the rat genome are unlikely.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer’s disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5′ region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11–p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

BACKGROUND Studies of genes that may inhibit growth or induce death of cells are facilitated greatly by tightly controlled expression of those genes. A promising system for control of transgene expression over a wide range is the tetracycline-repressible transactivator (tTA) system developed by Gossen and Bujard [Proc Natl Acad Sci USA 1992;89:5547–5551]. We investigated the effectiveness of this system in three well-established human prostate cancer cell lines. METHODS LNCaP, PC-3, and Tsu-Pr1 cells were transfected with a vector coding for the tTA protein and/or a luciferase reporter vector, and luciferase activity was measured in the presence and absence of tetracycline or the tTA protein. RESULTS In the absence of tetracycline, the tTA system yielded high levels of luciferase activity in all three cell lines. Background luciferase activity in the presence of tetracycline was nearly undetectable in LNCaP cells, moderate in Tsu-Pr1 cells, and more than 20-fold higher in PC-3 than in Tsu-Pr1 cells. Similar background activity was observed in Tsu-Pr1 and PC-3 cells, even in the absence of the transactivator protein. CONCLUSIONS The tTA system should be useful for stable transfection of cytotoxic transgenes in LNCaP cells and for control of transgene expression over a wide range in Tsu-Pr1 and PC-3 cells. Prostate 33:166–176, 1997. © 1997 Wiley-Liss, Inc.

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.

Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.

Human and pig cDNAs for a novel stomach protein, the product of a gene expressed at high levels specifically in cells of the antrum mucosa, have been characterized. The general exon/intron structure of the genomic DNA is conserved in humans and mice. The predicted protein sequences of the human and mouse mRNAs contain 185 and 184 amino acids, respectively. The protein isolated from pig antral extracts has an NH2 terminus consistent with cleavage of a 20-amino acid signal peptide. Human cDNA was expressed in E. coli to generate a protein antigen for antibody production. The antibodies detected polypeptides of ∼18 kDa in antrum extracts from all mammalian species tested. Immunocytochemistry located antrum mucosal protein (AMP)-18 to surface mucosal cells of the mouse antrum and, specifically, to secretion granules, suggesting that it is cosecreted with mucins. Antrum extracts and recombinant human AMP-18 exhibit growth-promoting activity on epithelial cells that can be blocked by the specific antisera. We suggest that AMP-18 is a “gastrokine” that maintains the integrity of the gastric mucosal epithelium.

The present study demonstrates that the murine keratinocyte cell line 3PC expresses the Ca(2+)-insensitive isoforms of protein kinase C (PKC) delta, epsilon, zeta and (at both the mRNA and protein levels), but does not express the Ca(2+)-sensitive PKC isoforms alpha, beta or gamma. Recombinant retroviral gene transduction was used to develop derivatives of this cell line that stably express high levels of 1 PKC beta I-related transcripts and proteins, and have increased levels of Ca(2+)-stimulated PKC enzyme activity. Functional overexpression of the PKC beta I isoform in 3PC cells enhances both 12-O-tetradecanoyl phorbol-13-acetate-induced growth inhibition, and Ca(2+)-induced morphologic differentiation.

By employing S1 nuclease mapping of total RNA isolated from porcine cerebral cortex, cerebellum, hypothalamus, pituitary, kidney, liver, pancreas, intestine, and antral mucosa, we have investigated gastrin gene expression in these tissues. Our results show that a gastrin gene is expressed only in the antral mucosal and pituitary tissues. Based on the amount of gastrin specific probe protected from S1 nuclease digestion in the presence of a given weight of total RNA, the amount of gastrin mRNA present in pituitary is approximately 330 times lower than in antral mucosa. These findings help establish the tissue distribution of gastrin gene expression.

The Fourth Paralogism is, in a sense, something of a stepchild, either passed over in silence or given a minimal treatment in any discussion of the Paralogisms proper. Among recent commentators, Jonathan Bennett cursorily dismisses the Fourth Paralogism in two quick paragraphs,' while Patricia Kitcher, in an article entitled "Kant's Paralogisms," refers to "all three paralogisms,"' and does not mention the fourth at all. This is done, of course, not without some justice: the Fourth Paralogism in A is more or less clearly directed toward refuting the thesis that we cannot be sure of the existence of the external world, and this task is shifted in B to the Refutation of Idealism; while in B the Fourth Paralogism appears to be reduced to a few remarks on what Sellars has called Kant's ignorabimus about the mind/body problem.3 It is not unreasonable to suggest that almost all the sustained treatments of the Fourth Paralogism have been undertaken within the context of discussing Kant's position on objectivity and his (putative) phenomenalism, and that qua Paralogism the Fourth is usually taken to be merely one of the more awkward of Kant's embarrassingly contrived tetrads. Nonetheless, there is a good deal of philosophical material in this Paralogism that goes beyond that of the Refutation of Idealism and that is directly relevant to Kant's view of the self. The purpose of this paper will be (i) to examine the two versions of the Fourth Paralogism, beginning with the formal presentation of the syllogism, in order to determine what Kant takes the Fourth Paralogism to be, (z) to suggest likely sources for this Paralogism, and finally, on the basis of this reading of the Fourth Paralogism, (3) to consider in what way the discussion of the Fourth Paralogism contributes to Kant's attack on "rational psychology." I hope also to establish an exegetical point, and one to which few of Kant's commentators subscribe: that, though the syllogisms