Pleomorphic Adenoma Gene 1 Expression Research Articles

PLAG1 g.8795C>T Mutation Regulates Early Body Weight in Hu Sheep by Weakening miR-139 Binding.

Sheep birth and weaning weights indicate their growth and survival. Thus, identifying molecular genetic markers for early body weight is important in sheep breeding. Pleomorphic adenoma gene 1 (PLAG1) is important for regulating birth weight and body length in mammals; however, its relationship with sheep body weight remains unknown. Here, the 3'-untranslated region (3'-UTR) of the Hu sheep PLAG1 gene was cloned, single nucleotide polymorphisms (SNPs) were screened, genotype-early body weight relationships were analyzed, and the possible molecular mechanism was explored. PLAG1 3'-UTR sequences with five forms of base sequences plus poly(A) tails were detected in Hu sheep and the g.8795C>T mutation was identified. Luciferase reporter assay indicated that the g.8795C>T mutation influenced PLAG1 post-transcriptional activity. miRBase prediction showed that the g.8795C>T mutation was located in the miR-139 seed sequence binding region, and miR-139 overexpression significantly decreased both PLAG1-CC and PLAG1-TT activities. Moreover, the luciferase activity of PLAG1-CC was significantly lower than that of the PLAG1-TT, but miR-139 inhibition substantially increased both PLAG1-CC and PLAG1-TT luciferase activities, suggesting that PLAG1 is the target gene of miR-139. Thus, the g.8795C>T mutation upregulates PLAG1 expression by weakening its binding with miR-139, promoting PLAG1 expression, and increasing Hu sheep birth and weaning weights.

PLAG1 silencing promotes cell chemosensitivity in ovarian cancer via the IGF2 signaling pathway.

Ovarian cancer (OC) is one of the most lethal gynecological diseases. Novel prognostic biomarkers and therapeutic targets for OC are urgently required. The aim of this study was to investigate the mechanisms that govern how pleomorphic adenoma gene 1 (PLAG1) influences the biological processes and chemosensitivity of OC cells via the insulin-like growth factor-2 (IGF2) signaling pathway. Differentially expressed genes in OC were selected based on bioinformatics data. OC and adjacent tissue specimen were collected, followed by the determination of the expression of PLAG1 and IGF2 signaling pathway-associated genes. The regulatory mechanisms of PLAG1 in OC cells were analyzed following treatment with pcDNA or small interfering RNA (siRNA), and included the assessment of cell proliferation, migration, invasion and cisplatin resistance. PLAG1 was identified as an upregulated gene in OC. OC tissues exhibited increased expression of PLAG1 and IGF2 compared with the controls. Moreover, PLAG1 was observed to positively regulate the IGF2 signaling pathway. The siRNA-mediated silencing of PLAG1 resulted in decreased expression of IGF2, IGF1 receptor and insulin receptor substrate 1, as well as inhibited proliferation, migration, invasion and cisplatin resistance of OC cells. Furthermore, the effect of PLAG1 was dependent on IGF2. PLAG1 may therefore be considered as a possible target for the treatment of OC.

The PLAG1 mRNA expression analysis among genetic variants and relevance to growth traits in Chinese cattle

Pleomorphic adenoma gene 1 (PLAG1) encodes a developmentally regulated zinc finger protein, locating in growth-related QTNs. The mRNA expression of this gene was investigated in different tissues and from two different developmental periods, whilst to explore the functions of PLAG1 in growth traits of cattle. The results showed that PLAG1 was expressed in all examined tissues. However, PLAG1 expression levels in all examined tissues were significantly different between the 5-month fetus and 36-month adult cattle. Our juvenile results indicated PLAG1 is primarily expressed in embryonic tissues of Chinese cattle. Furthermore, two variations were identified. Association analysis revealed that the two variations were associated with growth traits (p < 0.05 or p < 0.01). These new findings provide a comprehensive overview of the critical roles of PLAG1 in growth traits modulation and can be highlighted as candidate molecular markers in cattle breeding.

Abstract PR14: Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors

Abstract Mutations in microRNA processing genes arise in ~20% of Wilms tumors, the most common pediatric kidney cancer. These mutations block microRNA production, yet the key microRNA target genes that drive tumorigenesis in this setting are unknown. To address this question, we performed whole-transcriptome and small RNA sequencing in Wilms tumors. In tumors with microRNA processing mutations, the most significantly overexpressed gene was Pleomorphic adenoma gene 1 (PLAG1). PLAG1 has known oncogenic activity in other cancers but has never been studied in Wilms tumor. This transcription factor is normally expressed only in the developing kidney and liver, where it transactivates insulin-like growth factor 2 (IGF2). Because IGF2 overexpression plays a major role in Wilms tumor pathogenesis, we hypothesized that microRNA pathway mutations contribute to Wilms tumor formation by de-repressing PLAG1, thus overexpressing IGF2. To determine the effect of ectopic PLAG1 expression in vivo, we generated mice that overexpress PLAG1 throughout the developing kidney (Wt1-Cre; LSL-PLAG1). These mice develop grossly enlarged kidneys and die by 4 weeks of age. Prenatally, nephron production is impaired, resulting in fewer glomeruli and dilated tubules. Importantly, PLAG1-overexpressing kidneys progressively acquire several neoplastic characteristics. The kidney parenchyma becomes replaced by cysts that develop polyps and dysplastic thickening. As these dysplastic cells continue to proliferate and fill the cyst lumen, they form nodules that appear precancerous and exhibit markers of active proliferation (Ki-67) and Igf2 signaling (phospho-Erk1/2, phospho-S6). Thus, ectopic PLAG1 expression in the developing kidney recapitulates the two key features of Wilms tumor: blocked differentiation and neoplastic proliferation. We next confirmed that PLAG1 is a microRNA target gene that promotes proliferation of Wilms tumor cells in vitro. Luciferase reporter assays demonstrated that highly conserved sequences in the PLAG1 3’UTR can be targeted by miR-16 and miR-34a. These two miRNAs are strongly downregulated in Wilms tumors with microRNA processing mutations. In addition, mimics and inhibitors of miR-16 and miR-34a modulate endogenous PLAG1 and IGF2 expression in the Wilms tumor cell line WiT49. Finally, PLAG1 overexpression in WiT49 produced increased IGF2 expression, increased activating phosphorylation of its receptor, and increased proliferation. Lastly, we interrogated PLAG1 levels in publicly available data from the TARGET consortium. In 75 relapsed favorable-histology Wilms tumors, microRNA pathway mutations were seen in 14 cases. However, other genomic aberrations were also associated with high PLAG1 expression. For instance, chromosome 8p12, containing the PLAG1 locus, is amplified in 15 cases. Additionally, nine cases show loss of chromosome 1p, which includes the miR-34a locus. These copy number changes rarely overlap with each other or with microRNA pathway mutations. Together, however, these three changes account for 35 of the 75 tumors, and all 35 display high PLAG1 expression. Regardless of mechanism, high levels of PLAG1 correlated with earlier relapse, implying that high PLAG1 expression drives more aggressive tumors. In sum, Wilms tumors can produce high levels of the oncogenic transcription factor PLAG1 through 1p loss, 8p12 gain, or microRNA processing blockade. PLAG1 activates IGF2 transcription and enhances proliferation of Wilms tumor cells in vitro. Overexpression of PLAG1 in developing mouse kidneys produces impaired differentiation and neoplastic growth. These data establish PLAG1 as a novel mediator of Wilms tumor formation and a potential therapeutic target. Citation Format: Kenneth S. Chen, Emily K. Stroup, Albert Budhipramono, Joshua T. Mendell, James F. Amatruda. Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR14.

PLAG1 expression and target genes in the hypothalamo-pituitary system in male mice.

Knockout of pleomorphic adenoma gene 1 (PLAG1) in mice results in reduced fertility. To investigate whether PLAG1 is involved in reproductive control by the hypothalamo-pituitary system in males, we determined PLAG1 expression sites and compared gene expression between hypothalami and pituitary glands from Plag1 knockout and wildtype animals. Abundant expression of PLAG1 was detected throughout the pituitary gland, including gonadotropes and somatotropes. The hypothalamus also contained a large number of PLAG1-expressing cells. PLAG1 was expressed in some gonadotropin-releasing hormone neurons, but not in kisspeptin neurons. Gene ontology analysis indicated upregulation of cell proliferation in both structures, and of cholesterol biosynthesis in the hypothalamus, but functional confirmation is required. Expression levels of pituitary gonadotropins and gonadotropin-releasing hormone receptor, and of brain gonadotropin-releasing hormone and kisspeptin mRNA were unaffected in knockout mice. We conclude that PLAG1 deficiency does not have a major impact on the reproductive control by the hypothalamo-pituitary system.

Mutations in microRNA processing genes in Wilms tumors derepress the IGF2 regulator PLAG1.

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor pleomorphic adenoma gene 1 (PLAG1) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress PLAG1 through copy number alterations, and PLAG1 expression correlates with prognosis in Wilms tumors. PLAG1 overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, PLAG1 transactivates insulin-like growth factor 2 (IGF2), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

Circulating high mobility group AT-hook 2 and pleomorphic adenoma gene 1 in blood of patients with oral squamous cell carcinoma.

High mobility group AT-hook 2 (HMGA2) and pleomorphic adenoma gene 1(PLAG1) have been demonstrated to be elevated in many malignant tumors. However, the aim of this study was to evaluate HMGA2 and PLAG1 levels in blood as a non-invasive biomarker for oral squamous cell carcinoma (OSCC) diagnosis. qRT-PCR was performed to measure circulating HMGA2 and PLAG1 levels in OSCC patients (n=43) and matched cancer-free blood control group (n=21). Clinical data of all patients were recorded. Circulating HMGA2 and PLAG1 in the 43 OSCC patients was significantly higher than in control group (P<.001, P=.038, respectively). Furthermore, HMGA2 expression in OSCC patients with poor-moderate differentiation was increased compared with well-differentiated group. However, no significant differences in PLAG1 expression were detected when differentiation was considered. In addition, the receiver operating characteristic (ROC) curve analysis for circulating HMGA2 revealed an area under the ROC curve of 0.876 (95% confidence interval, 0.793-0.959; P<.001) with 65.1% sensitivity and 100% specificity in discriminating OSCC from controls at a cutoff value of 14.380, demonstrating significant diagnostic value for OSCC. Circulating HMGA2 levels are increased in OSCC patients and may potentially serve as a significant index to evaluate OSCC diagnosis.

Abstract 4662: Expression of PLAG1 in salivary duct carcinomas: its efficacy of the distinction of carcinoma ex pleomorphic adenoma type and de novo type

Abstract Salivary gland duct carcinoma (SDC) is one of the high-grade salivary gland carcinoma, classified into two patterns; i) carcinoma ex pleomorphic adenoma (Ca-ex-PA) type, which is derived from benign pleomorphic adenoma (PA), ii) de novo type, which is generated from normal salivary gland tissue. According to current WHO classification, Ca-ex-PA is diagnosed when pathological examination proves as follows, a) the mixture of the component of cancer cells and that of pre-existing PA cells, b) the existence of malignant cells after the resection of PA. However, current criteria cannot entirely be accurate from the viewpoint of disease onset. For example, i) if a benign component of PA is replaced completely by Ca-ex-PA, the Ca-ex-PA type can be misdiagnosed as de novo type because histopathological examination cannot clarify the existence of PA in its surgery specimen. Moreover, ii) if a de novo type SDC, arising from normal salivary tissue, is growing and infiltrate to the preexisting PA, the de novo type can be misdiagnosed as Ca-ex-PA type. Recently, Pleomorphic adenoma gene 1 (PLAG1) has been identified as the gene associated with tumorigenesis of PA. Subsequently, the overexpression of PLAG1 protein has been reported to be observed in both PAs and Ca-ex-PAs and useful for diagnosis of them. Thus, we considered that PLAG1 immunohistochemistry can enable us to distinguish Ca-ex-PA type and de novo type in SDCs more definitively. In this study, 23 SDC patients who underwent primary surgery for cancer at our institution were enrolled, including 14 Ca-ex-PA types and 9 de novo types. In each case, we had its formalin-fixed-paraffin-embedded tissue sample stained for PLAG1-antibodies (clone 3B7, Mouse mAb, 1:100, Abnova) and evaluated PLAG1 protein expression in the component of glandular-epithelial component and that of non-glandular-epithelial, respectively. PLAG1 immunostaining was scored as positive when the more than 10% nuclear staining of tumor cells are observed. In all 23 SDCs, there was no PLAG1 protein overexpression in glandular-epithelial component, irrespective of benign or malignant. In all 14 Ca-ex-PA types, positive findings of PLAG1 overexpression were observed in non-glandular-epithelial component. In all 9 de novo types, no non-glandular-epithelial component showed any signs of PLAG1 protein overexpression. There was no discrepancy of conventional criteria and the status of PLAG1, which are the cases; i) Ca-ex-PA which is misdiagnosed as de novo type, ii) de novo type which is misdiagnosed as Ca-ex-PA type. Conclusions : There is no difference between conventional criteria and PLAG1-based criteria in our cases. However, in the cases with obscure characteristics of PA morphologically, such as mucoid/myxoid or cartilaginous-like materials, findings of PLAG1-positive cells can help us to distinguish Ca-ex-PA type from de novo type more definitively. Citation Format: Takayoshi Suzuki, Satoshi Kano, Kanako Hatanaka, Yutaka Hatanaka, Tomohiro Sakashita, Takatsugu Mizumachi, Hiromitsu Hatakeyama, Akihiro Homma, Yoshihiro Matsuno, Satoshi Fukuda. Expression of PLAG1 in salivary duct carcinomas: its efficacy of the distinction of carcinoma ex pleomorphic adenoma type and de novo type [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4662. doi:10.1158/1538-7445.AM2017-4662

Expression and Clinical Significance of miR-26a and Pleomorphic Adenoma Gene 1 (PLAG1) in Invasive Pituitary Adenoma.

BackgroundAlthough pituitary adenoma is a malignant tumor, it can present as invasive growth in some cases. MicroRNA (miR)-26a has been found to be abnormally highly expressed in pituitary adenoma, indicating possible involvement in pathogenesis. As a known target gene of miR-26a, PLAG1 has abnormally low expression in pituitary adenoma. The correlation between miR-26a or PLAG1 expressional abnormality and occurrence of pituitary adenoma is still unknown, as is its association with invasiveness of pituitary adenoma.Material/MethodsPituitary adenoma tissues, including both invasive and non-invasive subtypes, were collected from our Neurosurgery Department, in parallel with normal pituitary tissues from postmortem autopsy. qRT-PCR was used to detect mRNA expression of miR-26a and PLAG1, while Western blotting was used to test PLAG1 protein expression. The correlation between miR-26a and PLAG1, and with pathological features, were analyzed. ROC analysis revealed the utility of miR-26a and PLAG1 in differential diagnosis of invasive/non-invasive pituitary tumors and in analyzing their effects on patient prognosis.ResultsMiR-26a was remarkably upregulated in pituitary tumors, while PLAG1 was downregulated, especially in invasive pituitary tumors. miR-26a and PLAG1 had higher diagnostic values for differentiating between invasive and non-invasive pituitary tumors (AUC=0.889 and 0.818, respectively). Those patients with miR-26 overexpression and PLAG1 downregulation had unfavorable prognosis. miR-26 and PLAG1 are independent factors affecting patient diagnosis.ConclusionsMiR-26a can facilitate occurrence of pituitary tumor and invasiveness, probably via inhibiting PLAG1 expression.

Loss of expression of Plag1 in malignant transformation from pleomorphic adenoma to carcinoma ex pleomorphic adenoma

PLAG1 (pleomorphic adenoma gene 1) is frequently activated in pleomorphic adenoma (PA). Carcinoma ex pleomorphic adenoma (CXPA) arises in PA, and PLAG1 expression is believed to be maintained from PA to CXPA, as it can contribute to the carcinogenesis process. To evaluate if PLAG1 is a good marker of malignant transformation from PA to CXPA as well as to evaluate if PLAG1 expression is associated with progression and histopathologic subtype of CXPA. Forty PAs, 21 residual PAs (without malignant transformation), and 40 CXPAs were analyzed by immunohistochemistry with PLAG1 antibody. The proportion of positive neoplastic cells was assessed according to a 2-tiered scale: >10% to 50%, and >50% positive cells. The CXPA group was classified according to histopathologic subtype and invasiveness degree. Thirty-seven PAs (92.5%), 15 residual PAs (71%), and 14 CXPAs (35%) were positive for PLAG1. In relation to the CXPA group, among the intracapsular cases, myoepithelial carcinoma and epithelial-myoepithelial carcinoma showed the highest level of PLAG1 expression. PLAG1 expression is lost when PA undergoes malignant transformation, possibly due to other pathway activation and different clone cells. In addition, PLAG1 expression seems to be present mainly in low-grade carcinomas and in cases with early phase of invasion, due to its regulation of oncogene-induced cell senescence. In CXPA, PLAG1 expression was most associated with myoepithelial differentiation. This way, loss of PLAG1 expression can be considered a hallmark of CXPA carcinogenesis, mainly when there is only epithelial differentiation.

PLAG1 expression is maintained in recurrent pleomorphic adenoma

The proto-oncogene (pleomorphic adenoma gene 1 (PLAG1)) is immunohistochemically overexpressed in pleomorphic adenoma (PA). Its expression in recurrent pleomorphic adenoma (RPA), however, has not been investigated. Since complex mechanisms are involved in tumor recurrence, the aim of this study was to investigate whether PLAG1 overexpression occurs in RPA. We studied PLAG1 protein expression in 40 PAs and 36 RPAs by immunohistochemistry. Cases with immunopositive cells were classified into two categories, between 10 and 50% and >50%. In both groups, PLAG1 expression was observed in both epithelial and myoepithelial cells. Of PAs, 37 cases (93%) were positive, while this was the case in 34 RPA cases (94%). Our findings suggest that in addition to morphological similarity, PA and RPA express PLAG1, which might play a role in tumor recurrence. Furthermore, as for PA, expression of PLAG1 can be considered a valuable diagnostic marker for RPA.

PLAG1 Expression From Pleomorphic Adenoma to Carcinoma Ex-Pleomorphic Adenoma

PLAG1 (pleomorphic adenoma gene 1) is a proto-oncogene located at 8q12 with tumor-suppressor gene function. It is frequently activated in pleomorphic adenoma (PA) of the salivary glands due to chromosomal aberrations, resulting in fusions with some genes. These phenomena results in transcriptional PLAG1 upregulated, and the …

Pleomorphic adenoma gene 1 mediates the role of karyopherin alpha 2 and has prognostic significance in hepatocellular carcinoma.

BackgroundKaryopherin alpha 2 (KPNA2) promotes tumor growth in hepatocellular carcinoma (HCC). We aimed to determine the content and clinical significance of mechanism underlying.MethodsThe association of transcriptional factor pleomorphic adenoma gene 1 (PLAG1) with KPNA2 was explored by co-immunoprecipitation. In vitro gain- and loss-of-function models were established to explore the functional interaction. Clinical samples from 314 HCC patients were applied to explore the clinical significance.ResultsWe found that PLAG1 could associate with KPNA2 and be promoted into nucleus by KPNA2. The increment of proliferative and metastatic abilities by KPNA2 over-expression can be significantly retarded by PLAG1 inhibition. The co-enrichment of KPNA2 and PLAG1 in nucleus is observed in clinical samples and can distinguish patients with the worst prognosis. The positive PLAG1 expression is an independent risk factor of recurrence free survival (HR: 1.766, 1.315-2.371; P = 0.000) and overall survival (HR: 1.589, 1.138-2.220; P = 0.007). Especially for patients with positive KPNA2 staining (N = 152), the positive PLAG1 expression is the sole risk factor for both recurrence free survival (HR: 1.749, 1.146-2.670; P = 0.010) and overall survival (HR: 1.662, 1.007-2.744; P = 0.047).ConclusionsThe nuclear import of PLAG1 by KPNA2 is essential for the role of KPNA2 in HCC cells and is significant to predict poor survival of HCC patients after hepatectomy.

Diagnostic Utility of PLAG1 Immunohistochemical Determination in Salivary Gland Tumors

PLAG1 (pleomorphic adenoma gene 1) is a proto-oncogene whose overexpression is a crucial oncogenic event in salivary gland pleomorphic adenomas (PA), and in carcinoma ex-PA. The aim of the present study is to evaluate the sensitivity and the specificity of PLAG1 as a marker in the differential diagnosis of salivary gland benign and malignant tumors. We examined 101 cases, including 36 PAs, 8 myoepitheliomas, 3 basal cell adenomas, and 1 canalicular adenoma among benign tumors; 16 mucoepidermoid carcinomas, 10 adenoid cystic carcinomas, 8 acinic cell carcinomas, 8 polymorphous low-grade adenocarcinomas, 7 salivary duct carcinoma, and 4 epithelial-myoepithelial carcinoma among malignant tumors. PLAG1 was diffusely positive in 94.4% of PAs and in all myoepitheliomas, although with a lower staining intensity. Among malignant tumors, 2 (25%) polymorphous low-grade adenocarcinomas and 1 salivary duct carcinoma ex-PA were positive. In conclusion, PLAG1 is a marker with good specificity for PA and could be a useful diagnostic adjunct in the diagnosis of salivary gland tumors. In particular, this marker is negative in the most common salivary carcinomas, including adenoid cystic carcinoma, mucoepidermoid carcinoma, and acinic cell carcinoma. However, some mimickers of PA, like polymorphous low-grade adenocarcinoma, may show occasional positivity for PLAG1, thus limiting its diagnostic use. In addition, carcinoma ex-PA shows consistent positivity, and therefore should be considered as a diagnostic possibility in case of a malignant tumor with PLAG1 expression.

Diagnostic utility of molecular and cytogenetic analysis in lipoblastoma: a study of two cases and review of the literature

Lipoblastoma is a benign neoplasm of embryonic white fat tissue that results from the proliferation of primitive adipocytes, in which histological features can be ambiguous. In order to discriminate between lipoblastoma and other lipogenic and lipomatous tumours, we studied chromosomal alterations and protein expression in two cases of lipoblastoma in infants. Standard cytogenetic analysis, fluorescence in-situ hybridization, array comparative genomic hybridization and Western blotting allowed us to demonstrate the presence of chromosome abnormalities involving the 8q11-13 region containing the pleomorphic adenoma gene 1 (PLAG1), which are classically reported in lipoblastoma, and aberrant expression of PLAG1. This report illustrates two different tumorigenic pathways implicating PLAG1 in lipoblastoma: amplification through multiple copies of a small marker chromosome derived from chromosome 8, and a paracentric inversion of the long arm of chromosome 8. Both these anomalies induced aberrant expression of PLAG1, emphasizing the role of PLAG1 in tumorigenesis. The aberrant expression of PLAG1 protein has been hypothesized, but this is the first report to demonstrate its occurrence in lipoblastoma.

Upregulation of Igf and Wnt signalling associated genes in pleomorphic adenomas of the salivary glands in PLAG1 transgenic mice

The Pleomorphic adenoma gene 1 (PLAG1) is involved in various human neoplasias, including pleomorphic adenomas of the salivary glands. Moreover, the oncogenic role of PLAG1 was clearly demonstrated in two independent PLAG1 transgenic mouse founders, in which PLAG1 expression could be targeted to different tissues using the Cre/loxP system. MMTV-Cre-mediated targeted overexpression of PLAG1 in the salivary glands of double transgenic offspring mice, referred to as P1-MCre and P2-MCre mice, induced pleomorphic adenomas in this organ. Igf2, a genuine PLAG1 target gene, was highly upregulated in those tumours as well as in human pleomorphic adenomas of the salivary glands. These and previous observations in other PLAG1-induced tumours e.g. breast adenomyoepitheliomas emphasize the importance of Igf upregulation in such tumours. In this study, further evidence for the role of Igf2 in PLAG1-induced tumourigenesis, is reported. Inactivation of Igf2 in P1-MCre mice leads to a significant delay in tumour development. Since tumour development is not fully abrogated by inactivation of Igf2, other signalling pathways are likely to contribute to PLAG1-induced tumourigenesis as well. Further studies revealed that several genes such as H19, Dlk1, Gtl2, Igfbp2, Igfbp3 and genes involved in Wnt signalling, such as Wnt6, Cyclin D1 and beta-catenin are upregulated in P1-MCre mice in which Igf2 is inactivated. In conclusion, we clearly demonstrate upregulation of several genes associated with Igf and Wnt signalling in PLAG1-induced pleomorphic adenomas. Furthermore, inactivation of Igf2 does not affect upregulation of genes associated with Wnt signalling, which might suggest that both signalling pathways are involved.

Sumoylation and Acetylation Play Opposite Roles in the Transactivation of PLAG1 and PLAGL2*

PLAG1 (pleomorphic adenoma gene 1) and PLAGL2 (PLAG-like 2) are oncogenes involved in various malignancies. Thus the study of their regulatory mechanisms may lead to identification of novel therapeutic targets. In this study, we provide supporting evidence that sumoylation and acetylation regulate functions of PLAG1 and PLAGL2. A conserved transcriptional repression domain exists in both PLAG1 and PLAGL2, whose activity depends on the presence of three sumoylation motifs and an intact sumoylation pathway. In vivo sumoylation assays confirmed that lysines 244, 263, and 353 of PLAG1 and lysines 250, 269, and 356 of PLAGL2 are indeed sumoylation sites. Further study showed that sumoylation inhibits PLAG1-induced IGF-II expression in reporter assays. The repression mediated by sumoylation may be partially explained by its effect on the cellular localization of PLAG1 and PLAGL2, because sumoylation-deficient but not wild-type PLAG1 and PLAGL2 concentrate in the nucleolus. PLAG1 and PLAGL2 are also regulated by acetylation. They are acetylated and activated by p300 and deacetylated and repressed by HDAC7. Interestingly, the sumoylation-deficient mutant of PLAGL2 is acetylated at a lower level than its wild-type counterpart, suggesting that some of the lysine residues may be targets for both modifications. Finally, mutation of three lysine residues in sumoylation motifs significantly impairs the transformation ability of PLAG1 and PLAGL2, suggesting the essential roles of these sites in the oncogenic potential of PLAG proteins. Taken together, the activities of PLAG1 and PLAGL2 are tightly modulated by both sumoylation and acetylation, which have opposite effects on their transactivation. To our knowledge, this is the first demonstration that oncoproteins can be regulated by both sumoylation and acetylation.

Repression of the Transactivating Capacity of the Oncoprotein PLAG1 by SUMOylation

Human pleomorphic adenoma gene 1 (PLAG1), a developmentally regulated proto-oncogene, is consistently rearranged and overexpressed in pleomorphic salivary gland adenomas and lipoblastomas with 8q12 translocations. Together with PLAGL1 and PLAGL2, PLAG1 belongs to a subfamily of C(2)H(2) zinc finger transcription factors that activate transcription through binding to the bipartite consensus sequence GRGGC(N)(6-8)GGG. Ectopic expression of PLAG1 deregulates target genes and presumably results in uncontrolled cell proliferation. To gain insight into molecular mechanisms regulating PLAG transcriptional capacity, we searched for interaction partners using the yeast two-hybrid system and confirmed these by glutathione S-transferase pull-down. Ubiquitin-conjugating enzyme 9 (UBC9) and protein inhibitor of activated STAT (PIAS) proteins were first identified as genuine interacting partners of mouse PlagL2. Because UBC9 and PIAS are components of the small ubiquitin-related modifier (SUMO) modification pathway, we hypothesized that PLAG proteins could be SUMOylated. Here, we report results obtained for founding family member PLAG1. Its endogenous SUMOylation was demonstrated, and SUMOylation of PLAG1 was further investigated in cells co-transfected with PLAG1 and SUMO-1 DNA or a SUMO-1 mutant form and similarly examined in the presence or absence of DNA encoding the various PIAS proteins. Using anti-PLAG1 antibodies, we discovered single and double SUMO-1-modified forms of PLAG1. By mutating predicted SUMO consensus sites, we defined two important target lysines for SUMOylation in PLAG1, Lys-244 and Lys-263. Moreover, mutation of both SUMO consensus sequences, resulting in inhibition of SUMOylation, led to a significant increase of the transactivation capacity of PLAG1. Nuclear distribution of PLAG1 was not measurably influenced. Our results suggest a direct repression of the transactivating capacity of the oncoprotein PLAG1 by SUMOylation.

Histologic Localization of PLAG1 (Pleomorphic Adenoma Gene 1) in Pleomorphic Adenoma of the Salivary Gland: Cytogenetic Evidence of Common Origin of Phenotypically Diverse Cells

Pleomorphic adenoma gene 1 (PLAG1), a zinc finger transcription factor gene, is consistently rearranged and overexpressed in human pleomorphic adenomas of the salivary glands with 8q12 translocations. In this report, we describe the immunohistochemical localization of PLAG1 protein in pleomorphic adenomas of the salivary gland and corresponding normal tissue, in relation to cytokeratin, vimentin, and BCL-2 expression. Normal salivary gland tissue was not immunoreactive for PLAG1. In primary pleomorphic adenomas, cells strongly immunoreactive for PLAG1 were detected in the outer layer of tubulo-ductal structures, which are thought to be the origin of cells with bi-directional, epithelial, and mesenchymal phenotypes. In contrast, epithelial cells with abundant cytokeratin in the inner tubulo-ductal structures only sporadically expressed PLAG1. BCL-2 immunoreactivity was found mainly in the cells surrounding the tubulo-ductal structures and in the solid undifferentiated cellular masses, within the areas that had moderate PLAG1 immunoreactivity. The variability of PLAG1 expression in neoplastic cells seemed to reflect the morphologic heterogeneity that correlated with the stage of differentiation of the tumor cells. Immunohistochemical/cytogenetic evaluation of two pleomorphic adenomas with t(3;8)(p21;q12) or t(5;8)(p13;q12) translocations demonstrated the clonal nature of immunophenotypically diverse cells. This finding confirms the theory that pleomorphic adenoma cells share a common single-cell origin, most likely from the epithelial progenitor basal duct cells.