The transcription factor nuclear factor-KB (NF-KB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-KB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-KB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-KB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-KB. Activation of TG2 led to activation of NF-KB; conversely, inhibition of TG2 activity inhibited activation of NF-KB. Similarly, ectopic expression of TG2 caused activation of NF-KB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-KB. Our results further indicated that constitutive NF-KB reporter activity in pancreatic cancer cells is not affected by dominant-negative IKBA. Additionally, coimmunoprecipitation and confocal microscopy showed that IKBA is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-KB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-KB in tumor cells via a novel pathway that is most likely independent of IKBA kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-KB activation and rendering cancer cells sensitive to anticancer therapies. (Cancer Res 2006; 66(17): 8788-95)

It is increasingly accepted that steroidal receptor coregulators may also function in the cytoplasmic compartment. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator that plays a role in both the genomic and extranuclear actions of estrogen receptors (ER) in hormonally responsive tissues. In this study using breast tumor arrays, we found that PELP1 was localized only in the cytoplasm in 58% of the PELP1-positive breast tumors. To help explain the significance of the cytoplasmic localization of PELP1 in human breast tumors, we created a mutant protein that was expressed only in the cytoplasm (PELP1-cyto) and then generated a model system wherein MCF-7 breast cancer cells were engineered to specifically express this mutant. We found that PELP1-cyto cells were hypersensitive to estrogen but resistant to tamoxifen. PELP1-cyto cells, but not parental MCF-7 cells, formed xenograft tumors in nude mice. In addition, PELP1-cyto cells exhibited increased association of PELP1 with Src, enhanced mitogen-activated protein kinase (MAPK) activation, and constitutive activation of AKT. The altered localization of PELP1 was sufficient to trigger the interaction of PELP1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K), leading to PI3K activation. In addition, PELP1 interacted with epidermal growth factor receptors and participated in growth factor-mediated ER transactivation functions. Our results suggest that the altered localization of PELP1 modulates sensitivity to antiestrogens, potentiates tumorigenicity, presumably via the stimulation of extranuclear estrogen responses, such as the activation of MAPK and AKT, and also enhance cross-regulation of ER transactivation activity by growth factors. (Cancer Res 2005; 65(17): 7724-32)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drugresistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C 277 S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC. (Cancer Res 2006; 66(21): 10525-33)

Regulation of fundamental genetic processes demands dynamic participation of transcription factors, their coregulators, and multiprotein chromatin remodeling activities at target genes. One family of chromatin modifiers that is ubiquitously expressed is the metastasis tumor antigens (MTA), which are integral parts of nucleosome remodeling and histone deacetylation (NuRD) complexes. MTA family members exist in distinct NuRD complexes, and functional redundancy is lacking among MTA family members. MTA proteins regulate divergent cellular pathways, including hormonal action, epithelial-to-mesenchymal transitions, differentiation, protein stability and development, and cell fate programs by modifying the acetylation status of crucial target genes. Intriguingly, at least one member of this family, MTA1, itself undergoes acetylation and acts as a coactivator in certain contexts. We discuss the roles of the MTA family of chromatin modifiers, with an emphasis on their physiologic functions.Dynamic alterations in chromatin structure facilitate or repress access of transcription complexes to target DNAs, leading to transcriptional changes and the regulation of functions associated with the gene products (1). These complexes modify DNA accessibility for cofactors by affecting DNA-histone interactions, nucleosome sliding, or relocation. In addition, the transcriptional state is influenced by covalent modification of the core histones (2). Among histone modifications, acetylation plays a pivotal role in chromatin remodeling (3). Core histones are subject to reversible acetylation at select lysine residues in their N-terminal domains through the coordinate activity of histone acetyltransferases and histone deacetylases (HDACs) 2 (3). Histone acetylation correlates with transcriptional activity, whereas deacetylation favors transcriptional repression. The mechanism of action of histone acetyltransferases is extensively reviewed elsewhere in the literature (4). Here we discuss the current understanding of the MTA family of coregulators, which are essential components of HDAC-containing NuRD transcriptional complexes. NuRD Complex, a Dynamic Switch for Histone Deacetylation and Chromatin RemodelingDeacetylation of histones via HDACs is carried out by two major complexes, Sin3 and NuRD (5, 6). The Sin3 complex contains seven polypeptides: HDAC1, HDAC2, RbAp46, RbAp48, Sin3, SAP18, and SAP30. This complex participates in nuclear hormone receptor repression of target genes (5, 7). NuRD complexes share four core proteins (HDAC1, HDAC2, RbAp46, and RbAp48) with the Sin3 complex and contain Mi-2␣/␤, MTA1/2, and p66 (5). In the NuRD complex, HDAC1/2 participate in the deacetylation process; Mi-2␣/␤ proteins with a chromodomain exhibit a DNA helicase/ATPase activity; and RbAp46/48 participate in histone binding. The roles of p66␣/␤ in NuRD complex are complex as p66 proteins are sumoylated, and SUMO-modified p66␣ efficiently interacts with HDAC1, whereas RbAp46 binds to SUMO-modified p66␤ (8, 9). All three MTA family proteins are found ...

Estrogen receptor ␣ (ER␣) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ER␣-interacting membranous and cytosolic proteins in ER␣ action, but our understanding of the role of the microtubule network in the modulation of ER␣ signaling remains unclear. Here we found that endogenous ER␣ associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ER␣-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ER␣, which in turn stimulates AKT and MAPK. We also found that ER␣ interacts with ␤-tubulin through HPIP. Destabilization of microtubules activated ER␣ signaling, whereas stabilization of microtubules repressed ER␣ transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIPmicrotubule complex in regulating 17␤-estradiol-ER␣ responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.17␤-estradiol ͉ estrogen receptor ͉ hematopoietic PBX-interaction protein E strogen regulates a plethora of functionally divergent physiological processes including development, homeostasis, and reproduction (1). The diversity of estrogen action results in part from the ability of estrogen receptors (ERs) to act both as transcription factors that regulate gene expression (i.e., genomic effects) and as signaling proteins that rapidly recruit and activate kinase-dependent signaling pathways (rapid effects). There is growing evidence that a subpopulation of the conventional nuclear steroid receptor localized in the vicinity of the cell membrane mediates many of the rapid signaling actions of steroid hormones; however, membrane receptors unrelated to conventional steroid receptors have also been implicated (2, 3). Several studies support the concept that estrogen can activate multiple cytosolic signaling pathways through direct interactions of conventional estrogen receptor (ER␣ or ER␤) with various cytoplasmic and membranous proteins, including kinases and adaptor proteins, by forming different multiprotein complexes (2, 4). In addition, sequestration of ER by MTA1s (metastasisassociated antigen 1 short form) also triggers estrogen rapid signaling. So it appears that relative subcellular distribution of ERs plays a critical role in estrogen signaling. Besides these mechanistic studies, recent reports have suggested that extranuclear estrogen signaling is directly implicated in cell migration through actin cytoskeleton remodeling (5).Microtubules are structural components of the cytoskeleton required for cell motility that regulate a variety of signaling pathways, including the inducible nitric oxide synthase, NF-B, ERK, JNK, Wnt, and Hedgehog signaling pathways (6). The functional role of microtubules in signal transduction has been further elucidated ...

Of late, nimbolide, a limonoid from the neem tree (Azadirachta indica) has gained increasing research attention owing to its potent antiproliferative and apoptosis-inducing effects. The present study was designed to investigate the effect of nimbolide on autophagy and the time point at which the phosphorylation status of GSK-3β and PI3K dictate the choice between autophagy and apoptosis in SCC131 and SCC4 oral cancer cells. Additionally, we analysed changes in the expression of proteins involved in autophagy and apoptosis after therapeutic intervention with nimbolide in a hamster model of oral oncogenesis. Furthermore, we also demonstrate changes in the expression of key genes involved in apoptosis and autophagy during the stepwise evolution of hamster and human OSCCs. Nimbolide-induced stereotypical changes in oral cancer cells characteristic of both apoptosis and autophagy. Time-course experiments revealed that nimbolide induces autophagy as an early event and then switches over to apoptosis. Nimbolide negatively regulates PI3K/Akt signalling with consequent increase in p-GSK-3βTyr216, the active form of GSK-3β that inhibits autophagy. Downregulation of HOTAIR, a competing endogenous RNA that sponges miR-126 may be a major contributor to the inactivation of PI3K/Akt/GSK3 signalling by nimbolide. Analysis of key markers of apoptosis and autophagy as well as p-AktSer473 during sequential progression of hamster and human OSCC revealed a gradual evolution to a pro-autophagic and antiapoptotic phenotype that could confer a survival advantage to tumors. In summary, the results of the present study provide insights into the molecular mechanisms by which nimbolide augments apoptosis by overcoming the shielding effects of cytoprotective autophagy through modulation of the phosphorylation status of Akt and GSK-3β as well as the ncRNAs miR-126 and HOTAIR. Development of phytochemicals such as nimbolide that target the complex interaction between proteins and ncRNAs that regulate the autophagy/apoptosis flux is of paramount importance in cancer prevention and therapeutics.

Estrogen mediate its biological effects through its association with estrogen receptors (ERs). They also regulate the expression of a variety of genes involved in distinct physiological processes, including development, metabolism, and reproduction. In addition, emerging data suggest that the estrogen-estrogen receptor complex can also function as a cytoplasmic signaling molecule and may influence processes such as cardiovascular protection, bone preservation, neuroprotection, and proliferation of various cell types. Such extranuclear or nongenomic signaling pathways are rapid and supposedly independent of transcription. A recent exciting finding was that G-coupled membrane protein receptor, GPR30, an alternative to the classical ERs, is also involved in the rapid signaling of estrogen through its direct association with estrogen. These new findings combined with the recent advances in the cytoplasmic functions of proline, glutamic acid, luecine rich protein 1 (PELP1), and metastatic tumor antigen 1 short form (MTA1s) have opened a new spectrum and raised several new concerns in the field of estrogen biology and put the attention to unveil many unknown mechanistic actions of estrogen in cellular physiology. In this review, we briefly summarize what is currently known of the cellular mechanisms and physiology of estrogen's nongenomic actions in various cellular systems used by ERs.

IntroductionMetastasis is a complex disease involving a series of events that includes stimulation or repression of numerous gene products in a coordinate manner, resulting in the invasion of neoplastic cells and detachment from the primary tumors, penetration into blood and lymphatics, arrest by adhesion at distant sites, extravasation, induction of angiogenesis, evasion of host antitumor responses, and growth at metastatic sites [Nicolson, 1988]. To identify the genes potentially involved in breast cancer invasion and metastasis, Toh et al. performed differential screening of a cDNA library of metastatic and nonmetastatic adenocarcinoma cell lines from rat mammary glands and cloned a novel gene known as the metastasis-associated tumor gene (mta1) [Toh et al., 1994]. Despite widespread overexpression of the MTA1 protein in human tumors, the molecular functions of the MTA family remained a mystery until the proteomic analysis of the nucleosome remodeling and deacetylation (NuRD) complex identified MTA1 and MTA2 as integral to the histone deacetylase (HDAC) complexes [Xue et al., 1998;Zhang et al., 1998], thus providing clues about the role of the MTA family of proteins in chromatin remodeling. MTA3, the third member of the MTA family, was identified as an estrogen-inducible gene product that forms a distinct Mi-2/NuRD complex [Fujita et al., 2003]. MTA1s, a naturally occurring variant of MTA1, was accidentally discovered by researchers pursuing the role of MTA1 in transcriptional regulation of estrogen receptor-α (ER) [Kumar et al., 2002]. MTA1s is generated by alternative splicing at a cryptic splice-site in exon 14, involving the deletion of 47 base pair nucleotides, and a frame-shift involving the addition of 33 unique amino acids with no homology among sequences from Genbank. The resulting MTA1s gene product lacks the C-terminal region of other MTA family members [Kumar et al., 2002]. Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas, researchers identified ZG29p, a cDNA coding for the 29-kDa protein, by immunoscreening a hormonally stimulated pancreas cDNA library. ZG29p is an N-terminal truncated form of MTA1, coded by the last seven exons of MTA1 and present in the zymogen granules of the pancreas. ZG29p mediates an interaction with amylase and is involved in condensation-sorting in the exocrine rat pancreas [Kleene et al., 2000]. Domain structure of the MTA familyMTA1 and MTA2 are polypeptides with molecular masses of approximately 80 kDa and 70 kDa, respectively, whereas MTA3 is smaller, approximately 65 kDa. Protein alignment homology of the human MTA proteins revealed 68% and 73% homology of MTA2 and MTA3, respectively, with MTA1. The highest homology was concentrated in the N-terminal half of the proteins, whereas the C-terminal regions of the MTA proteins were divergent (Figure 1A and [Manavathi and Kumar, 2007] has not yet been directly tested; it is currently hypothesized from studies of proteins containing such domains. For example, the BAH domain ...