The High Mobility Group A1 (HMGA1) gene is highly overexpressed in human uterine serous carcinomas and carcinosarcomas and drives Matrix Metalloproteinase-2 (MMP-2) in a subset of tumors.

OBJECTIVES: Although uterine cancer is the fourth most common cause for cancer death in women worldwide, the molecular underpinnings of tumor progression remain poorly understood. The High Mobility Group A1 (HMGA1) gene is overexpressed in aggressive cancers and high levels portend adverse outcomes in diverse tumors. We previously reported that Hmga1a transgenic mice develop uterine tumors with complete penetrance. Because HMGA1 drives tumor progression by inducing MatrixMetalloproteinase (MMP) and other genes involved in invasion, we explored the HMGA1-MMP-2 pathway in uterine cancer. METHODS: To investigate MMP-2 in uterine tumors driven by HMGA1, we used a genetic approach with mouse models. Next, we assessed HMGA1 and MMP-2 expression in primary human uterine tumors, including low-grade carcinomas (endometrial endometrioid) and more aggressive tumors (endometrial serous carcinomas, uterine carcinosarcomas/malignant mesodermal mixed tumors). RESULTS: Here, we report for the first time that uterine tumor growth is impaired in Hmga1a transgenic mice crossed on to an Mmp-2 deficient background. In human tumors, we discovered that HMGA1 is highest in aggressive carcinosarcomas and serous carcinomas, with lower levels in the more indolent endometrioid carcinomas. Moreover, HMGA1 and MMP-2 were positively correlated, but only in a subset of carcinosarcomas. HMGA1 also occupies the MMP-2 promoter in human carcinosarcoma cells. CONCLUSIONS: Together, our studies define a novel HMGA1-MMP-2 pathway involved in a subset of human carcinosarcomas and tumor progression in murine models. Our work also suggests that targeting HMGA1 could be effective adjuvant therapy for more aggressive uterine cancers and provides compelling data for further preclinical studies.

The HMGA1-COX-2 axis: a key molecular pathway and potential target in pancreatic adenocarcinoma.

CONTEXT: Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions. METHODS: Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis. RESULTS: HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1. CONCLUSIONS: Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.

HMGA1 correlates with advanced tumor grade and decreased survival in pancreatic ductal adenocarcinoma.

Although pancreatic ductal adenocarcinoma is a common and almost uniformly fatal cancer, little is known about the molecular events that lead to tumor progression. The high-mobility group A1 (HMGA1) protein is an architectural transcription factor that has been implicated in the pathogenesis and progression of diverse human cancers, including pancreatic ductal adenocarcinoma. In this study, we investigated HMGA1 expression in pancreatic ductal adenocarcinoma cell lines and surgically resected tumors to determine whether it could be a marker for more advanced disease. By real-time quantitative RT-PCR, we measured HMGA1a mRNA in cultured pancreatic ductal adenocarcinoma cell lines and found increased levels in all cancer cells compared with normal pancreatic tissue. To investigate HMGA1 in primary human tumors, we performed immunohistochemical analysis of 125 cases of pancreatic adenocarcinoma and 99 precursor lesions (PanIN 1-3). We found nuclear staining for HMGA1 in 98% of cases of pancreatic adenocarcinoma, but only 43% of cases of PanIN precursor lesions. Moreover, HMGA1 immunoreactivity correlates positively with decreased survival and advanced tumor and PanIN grade. These results suggest that HMGA1 promotes tumor progression in pancreatic ductal adenocarcinoma and could be a useful biomarker and rational therapeutic target in advanced disease.

Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health.

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.

HMGA2 participates in transformation in human lung cancer.

Although previous studies have established a prominent role for HMGA1 (formerly HMG-I/Y) in aggressive human cancers, the role of HMGA2 (formerly HMGI-C) in malignant transformation has not been clearly defined. The HMGA gene family includes HMGA1, which encodes the HMGA1a and HMGA1b protein isoforms, and HMGA2, which encodes HMGA2. These chromatin-binding proteins function in transcriptional regulation and recent studies also suggest a role in cellular senescence. HMGA1 proteins also appear to participate in cell cycle regulation and malignant transformation, whereas HMGA2 has been implicated primarily in the pathogenesis of benign, mesenchymal tumors. Here, we show that overexpression of HMGA2 leads to a transformed phenotype in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA2 expression blocks the transformed phenotype in metastatic human non-small cell lung cancer cells. Moreover, we show that HMGA2 mRNA and protein are overexpressed in primary human lung cancers compared with normal tissue or indolent tumors. In addition, there is a statistically significant correlation between HMGA2 protein staining by immunohistochemical analysis and tumor grade (P < 0.001). Our results indicate that HMGA2 is an oncogene important in the pathogenesis of human lung cancer. Although additional studies with animal models are needed, these findings suggest that targeting HMGA2 could be therapeutically beneficial in lung cancer and other cancers characterized by increased HMGA2 expression.

The high-mobility group A1 gene up-regulates cyclooxygenase 2 expression in uterine tumorigenesis.

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers.

Cyclooxygenase inhibitors block uterine tumorigenesis in HMGA1a transgenic mice and human xenografts.

Uterine cancer is a common cause for cancer death in women and there is no effective therapy for metastatic disease. Thus, research is urgently needed to identify new therapeutic agents. We showed previously that all female HMGA1a transgenic mice develop malignant uterine tumors, indicating that HMGA1a causes uterine cancer in vivo. We also demonstrated that HMGA1a up-regulates cyclooxygenase-2 (COX-2) during tumorigenesis in this model. Similarly, we found that HMGA1a and COX-2 are overexpressed in human leiomyosarcomas, a highly malignant uterine cancer. Although epidemiologic studies indicate that individuals who take COX inhibitors have a lower incidence of some tumors, these inhibitors have not been evaluated in uterine cancer. Here, we show that HMGA1a mice on sulindac (a COX-1/COX-2 inhibitor) have significantly smaller uterine tumors than controls. To determine if COX inhibitors are active in human uterine cancers that overexpress HMGA1a, we treated cultured cells with sulindac sulfide or celecoxib (a specific COX-2 inhibitor). Both drugs block anchorage-independent growth in high-grade human uterine cancer cells that overexpress HMGA1a (MES-SA cells). In contrast, neither inhibitor blocked transformation in cells that do not overexpress HMGA1a. Moreover, xenograft tumors from MES-SA cells were significantly inhibited in mice on sulindac. More strikingly, no tumors formed in mice on celecoxib. These preclinical studies suggest that COX inhibitors could play a role in preventing tumor onset or progression in uterine cancers with dysregulation of the HMGA1a-COX-2 pathway. Importantly, these drugs have lower toxicity than chemotherapeutic agents used to treat advanced-stage uterine cancers.

Involvement of Akt in preconditioning-induced tolerance to ischemia in PC12 cells.

The serine-threonine protein kinase Akt has been identified as an important mediator of cell survival able to counteract apoptotic stimuli. However, hibernation, a model of natural tolerance to cerebral ischemia, is associated with downregulation of Akt. We previously established a model of ischemic tolerance in a PC12 cell line and using this model we now addressed the question whether ischemic tolerance also downregulates Akt in PC12 cells. Kinetic studies showed decreased Akt phosphorylation in tolerized cells. Similarly, phosphorylated levels of three major targets of Akt and well-known proapoptotic factors, the glycogen synthase kinase 3 (GSK-3), a Forkhead family member, FoxO4, and the protein murine double minute 2 (MDM2), all inactivated upon phosphorylation by Akt, were decreased in preconditioned cells. In addition, pharmacological blockade of the phosphoinositide 3-kinase (PI3K)/Akt pathway reduced cell death induced by oxygen and glucose deprivation (OGD) and increased the protective effect of preconditioning (PC). Furthermore, decreasing availability of P-Akt by transfecting PC12 cells with constructs of inactive Akt also resulted in protection against OGD and potentiation of the protective effect of PC. Depending on the environment, GSK-3, FOXO-4, and MDM2 can trigger apoptotic responses or cell cycle arrest, and thus, in a situation of reduced energy, driving the cells into a state of quiescence might be neuroprotective. This work suggests that in the context of tolerance downregulation of Akt is beneficial.

Development of an ischemic tolerance model in a PC12 cell line.

Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia.

Adenosine A2A-dopamine D2 receptor-receptor heteromers. Targets for neuro-psychiatric disorders.

Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D(2) receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D(2) receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson's disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D(2) receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D(2) receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D(2) receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D(2) receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction.

Inactivation of the Cdkn2a locus cooperates with HMGA1 to drive T-cell leukemogenesis.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia with high relapse rates compared to B-lineage ALL. We previously showed that HMGA1a transgenic mice develop aggressive T-ALL, indicating that HMGA1 causes leukemic transformation in vivo. HMGA1 is also highly expressed in embryonic stem cells, hematopoietic stem cells and diverse, refractory human cancers. Disruption of the CDKN2A tumor suppressor locus occurs in most cases of T-ALL and is thought to contribute to leukemic transformation. To determine whether loss of function of CDKN2A cooperates with HMGA1 in T-ALL, we crossed HMGA1a transgenics onto a Cdkn2a null background. We discovered that T-ALL is markedly accelerated in HMGA1a transgenic Cdkn2a null mice. In addition, these mice recapitulate salient clinical and pathologic features of human T-ALL. HMGA1 is also highly overexpressed in human T-ALL. These findings suggest that HMGA1 plays a causative role in T-ALL and could represent a rational therapeutic target.

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks.

BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

Flavopiridol induces BCL-2 expression and represses oncogenic transcription factors in leukemic blasts from adults with refractory acute myeloid leukemia.

Flavopiridol is a cyclin-dependent kinase inhibitor that induces cell cycle arrest, apoptosis, and clinical responses in selected patients with acute myeloid leukemia (AML). A better understanding of the molecular pathways targeted by flavopiridol is needed to design optimal combinatorial therapy. Here, we report that in vivo administration of flavopiridol induced expression of the BCL-2 anti-apoptotic gene in leukemic blasts from adult patients with refractory AML. Moreover, flavopiridol repressed the expression of genes encoding oncogenic transcription factors (HMGA1, STAT3, E2F1) and the major subunit of RNA Polymerase II. Our results provide mechanistic insight into the cellular pathways targeted by flavopiridol. Although further studies are needed, our findings also suggest that blocking anti-apoptotic pathways could enhance cytotoxicity with flavopiridol.

Upregulation of MMP-2 by HMGA1 promotes transformation in undifferentiated, large-cell lung cancer.

Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1-MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors.

The high-mobility group A1a/signal transducer and activator of transcription-3 axis: an achilles heel for hematopoietic malignancies?

Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.

Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures.

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.

Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors and dopamine D2 receptors.

Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A(2A) receptors (A(2A)R) and dopamine D(2) receptors (D(2)R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D(2)R and in cultured striatal cells. A(2A)R/D(2)R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D(2)R-transfected SH-SY5Y cells and from mouse fibroblast Ltk(-) cells stably transfected with human D(2)R (long form) and transiently cotransfected with the A(2A)R double-tagged with hemagglutinin. Long term exposure to A(2A)R and D(2)R agonists in D(2)R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A(2A)R and D(2)R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D(2)R are involved.