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PP-1α and PP-1γ Display Antagonism and Differential Roles in Tumorigenicity of Lung Cancer Cells
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Protein serine/threonine phosphatases are important cellular signaling molecules and play major roles in regulating many different functions including cell proliferation, senescence, programmed cell death, and oncogenic cell transformation. Among different serine/threonine phosphatases, PP-1 and PP-2A contribute to more than 90% phosphatase activities in eukaryotes. While the functions of PP-2A in cell transformation and tumorigenesis have been well established, the role of PP-1 in carcinogenesis remains to be further explored. Moreover, PP-1 exists in different isoforms, whether these isoforms have differential functions in tumorigenesis remains to be examined. In the present study, we demonstrated that in lung cancer 1299 cells, PP1α and PP-1γ exist in an antagonizing balance. In the parent H1299 cells, PP-1γ is dominant, about 4-fold higher than that of PP-1. Overexpression of PP-1α significantly down-regulates PP-1γ at both mRNA and protein levels. In contrast, knockdown of PP-1α leads to upregulation of PP-1γ. Moreover, overexpression of PP-1α significantly attenuates the ability of the H1299 cells in promoting tumorigenecity as tested in immuno-deficient nude mice. This attenuation is derived from the halted cell cycle progression, which is largely attributed by the changed RB-E2F activity. Together, our results demonstrate that PP-1α and PP-1γ not only antagonize each other in lung cancer cells, but also display differential functions in tumorigenecity.
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Novel and accurate biomarkers are needed for early detection and progression evaluation of hepatocellular carcinoma (HCC). Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) has been studied in cancer biology; however, the expression pattern and biological function of PPP1R1A in HCC are unclear. The differentially expressed genes (DEGs) in HCC were screened by The Cancer Genome Atlas (TCGA) database. Real-time PCR and immunohistochemistry (IHC) assay were used to detect the expression of PPP1R1A in BALB/c mice, human normal tissues and corresponding tumor tissues, especially HCC. Then, Kaplan–Meier analysis of patients with HCC was performed to evaluate the relationship between PPP1R1A expression and prognosis. The transcriptional regulatory network of PPP1R1A was constructed based on the differentially expressed mRNAs, microRNAs and transcription factors (TFs). To explore the downstream regulation of PPP1R1A, the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis and immune infiltration score were performed. A total of 4 DEGs were screened out. PPP1R1A was differentially distributed and expressed in BALB/c mice and human tissues. PPP1R1A expression was higher in normal tissues than that in tumor tissues, and patients with higher PPP1R1A expression had better clinical outcome in HCC. In addition, we constructed miR-21-3p/TAL1/PPP1R1A transcriptional network. Furthermore, PPP1R1A may modulate the activation of PI3K–Akt pathway, cell cycle, glycogen metabolism and the recruitment of M2 macrophage in HCC. This study may help to clarify the function and mechanism of PPP1R1A in HCC and provide a potential biomarker for tumor prevention and treatment.
The Ser/Thr-protein phosphatase PP1 (PP1) is a positive regulator of the androgen receptor (AR), which suggests major roles for PP1 in prostate carcinogenesis. However, studies dedicated to the characterization of PP1 in PCa are currently scarce. Here we analyzed the expression and localization of the PP1 catalytic (PP1c) isoforms in formalin-fixed, paraffin-embedded prostate tissue samples, as well as in PCa cell lines. We also analyzed well-characterized PCa cohorts to determine their transcript levels, identify genetic alterations, and assess promoter methylation of PP1c-coding genes. We found that PP-1A was upregulated and relocalized towards the nucleus in PCa and that PPP1CA was frequently amplified PCa, particularly in advanced stages. PP-1B was downregulated in PCa but upregulated in a subset of tumors with AR amplification. PP-1G transcript levels were found to be associated with Gleason score. PP1c-coding genes were rarely mutated in PCa and were not prone to regulation by promoter methylation. Protein phosphorylation, on the other hand, might be an important regulatory mechanism of PP1c isoforms' activity. Altogether, our results suggest differential expression, localization, and regulation of PP1c isoforms in PCa and support the need for investigating isoform-specific roles in prostate carcinogenesis in future studies.
Cancer cells take advantage of signaling cascades to meet their requirements for sustained growth and survival. Cell signaling is tightly controlled by reversible protein phosphorylation mechanisms, which require the counterbalanced action of protein kinases and protein phosphatases. Imbalances on this system are associated with cancer development and progression. Protein phosphatase 1 (PP1) is one of the most relevant protein phosphatases in eukaryotic cells. Despite the widely recognized involvement of PP1 in key biological processes, both in health and disease, its relevance in cancer has been largely neglected. Here, we provide compelling evidence that support major roles for PP1 in tumorigenesis.
Our recent study has shown that αA-crystallin appears to act as a tumor suppressor in pancreas. Here, we analyzed expression patterns of αA-crystallin in the pancreatic tumor tissue and the neighbor normal tissue from 74 pancreatic cancer patients and also pancreatic cancer cell lines. Immunocytochemistry revealed that αA-crystallin was highly expressed in the normal tissue from 56 patients, but barely detectable in the pancreatic tumor tissue. Moreover, a low level of αA-crystallin predicts poor prognosis for patients with pancreatic duct adenocarcinoma (PDAC). In the 12 pancreatic cell lines analyzed, except for Capan-1 and Miapaca-2 where the level of αA-crystallin was about 80% and 65% of that in the control cell line, HPNE, the remaining pancreatic cancer cells have much lower αA-crystallin levels. Overexpression of αA-crystallin in MiaPaca-1 cells lacking endogenous αA-crystallin significantly decreased its tumorigenicity ability as shown in the colony formation and wound healing assays. In contrast, knockdown of αA-crystallin in the Capan-1 cells significantly increased its tumorigenicity ability as demonstrated in the above assays. Together, our results further demonstrate that αA-crystallin negatively regulates pancreatic tumorigenesis and appears to be a prognosis biomarker for PDAC.
The A subunit of protein phosphatase 2A (PP2A) consists of 15 nonidentical repeats. The catalytic C subunit binds to C-terminal repeats 11–15 and regulatory B subunits bind to N-terminal repeats 1–10. Recently, four cancer-associated mutants of the Aα subunit have been described: Glu64→Asp in lung carcinoma, Glu64→Gly in breast carcinoma, Arg418→Trp in melanoma, and Δ171–589 in breast carcinoma. Based on our model of PP2A, we predicted that Glu64→Asp and Glu64→Gly might be defective in B subunit binding, whereas Arg418→Trp and Δ171–589 might bind neither B nor C subunits. We generated these mutants by site-directed mutagenesis and assayed their ability to associate with different forms of B subunits (B, B′, B′′) or with the catalytic C subunit. The results demonstrate that all mutants are defective in binding either B or B and C subunits. Specifically, the N-terminal mutants, Glu64→Asp and Glu64→Gly, are defective in B′ but normal in B, B′′, and C subunit binding, whereas the C-terminal mutants Arg418→Trp and Δ171–589 bind none of the B subunits nor the C subunit. The implications of these findings with regard to the potential role of PP2A as a tumor suppressor are discussed.
The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the β isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both α (PPP2R1A) and β isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.
Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, a , g 1, and d , have nearly identical catalytic domains, but they vary in sequence at their extreme NH 2 and COOH ter- mini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mam- malian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 a associates with the nuclear matrix, PP-1 g 1 con- centrates in nucleoli in association with RNA, and PP-1 d localizes to nonnucleolar whole chromatin. During mitosis, PP-1 a is localized to the centrosome, PP-1 g 1 is associated with microtubules of the mitotic spindle, and PP-1 d strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.
Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection
of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human
cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation
at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data
provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that
asbestos and SV40 may be cocarcinogens.
Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation–dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles.
Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5+ gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.
PP2A plays a critical role in growth control and cancer. Importantly, loss or alteration of PP2A activity is an essential step in the development of human cancer, consistent with the idea that PP2A functions as a tumor suppressor. However, PP2A has many, sometimes seemingly conflicting, functions that are poorly understood. On the one hand, it suppresses cell growth, but on the other it is required for cell-cycle progression. Also, it positively and negatively regulates the MAPK/ERK and Wnt signaling pathways. Other important functions of PP2A are its inhibitory role in the interleukin-3-stimulated JAK2-STAT5 signaling pathway and its involvement in NF-KB signaling, protein kinase B/Akt signahng, and integrin-mediated regulation of Akt and GSKSP. Furthermore, the catalytic subunit of PP2A binds to the alpha-4 protein, a homolog of yeast TAP42 involved in translational control.
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.
Complementary DNA encoding a catalytic subunit of protein phosphatase 1, termed PP1β, was isolated from a human teratocarcinoma library. Hybridisation with different cDNA fragments showed that all human tissues examined contained 3.1 kb, 4.0 kb and 5.4 kb PP1β mRNAs arising from alternative splicing of the 3′ noncoding region. The level of the 5.4 kb mRNA relative to the 3.1 kb mRNA was higher in skeletal muscle than in other tissues and the mRNA ratio in rabbit tissues was highest in skeletal muscle. The 3′ noncoding region of PP1β showed extreme conservation (≥ 90% identity) between man and rodents over 1.7 kb, suggesting that this region is of functional importance. The gene for human PP1β (PPP1CB) was localised to chromosome 2 by analysis of somatic cell hybrid DNA and mapped to band q23 by fluorescence in situ hybridisation. These data show that the genes for three protein phosphatase catalytic subunits PP1α, PP1β, PP1γ are all located on different chromosomes.