Amy Belton

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.

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.

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. (Mol Cancer Res 2009;7(11):1803–12)

HMGA1 overexpression correlates with relapse in childhood B-lineage acute lymphoblastic leukemia

Despite dramatic improvements in treatment and survival, acute lymphoblastic leukemic (ALL) remains the leading cause of death in children with cancer [1]. ALL is the most common pediatric cancer, accounting for 25% of all cancers occurring in children under the age of 15 years [1]. The majority of cases are a B-lineage cell subtype of ALL (B-ALL). Although most children with ALL are cured with current chemotherapeutic regimens, approximately 15% will relapse and 8–9% will ultimately succumb to their disease [1]. Risk assessment is a critical component in the selection of appropriate therapy for individual patients. Unfortunately, current criteria used to stratify patients into groups at increasing risk for treatment failure or relapse are limited, and rely primarily on clinical characteristics (age, white blood cell count, immunophenotype, presence/absence of central nervous system disease or testicular disease) and underlying genetic lesions. Good risk genetic lesions include ETV6–RUNX1 fusion or hyperdiploidy (50 or more chromosomes) with favorable chromosomal trisomies (4 + 10), while poor risk genetic lesions include MLL rearrangements (MLL-R), hypodiploidy, intrachromosome 21 (iAMP21) or Philadelphia chromosome positive (Ph+) ALL [1]. In addition, early treatment response is included in risk stratification and determined by measurement of minimal residual disease (MRD) at specific time points early in therapy. The 5-year overall survival in ALL has increased from only 10% in the 1960s to about 80% in the 1980s, and very recent studies indicate that 92% of children with ALL will be cured with current therapies [1,2]. Nonetheless, many patients are over-treated, whereas a significant number of children who were initially classified as favorable risk suffer a relapse. Thus, elucidating the molecular underpinnings of refractory disease in ALL is needed to identify patients who are likely to relapse and require more intensive chemotherapeutic regimens. Moreover, identifying key molecular pathways could also uncover novel therapeutic targets.

SOD2, the principal scavenger of mitochondrial superoxide, is dispensable for embryogenesis and imaginal tissue development but essential for adult survival

Definitive evidence on the impact of MnSOD/SOD2-deficiency and the consequent effects of high flux of mitochondrial reactive oxygen species (ROS) on pre-natal/pre-adult development has yet to be reported for either Drosophila or mice. Here we report that oocytes lacking maternal SOD2 protein develop into adults just like normal SOD2-containing oocytes suggesting that maternal SOD2-mediated protection against mitochondrial ROS is not essential for oocyte viability. However, the capacity of SOD2-null larvae to undergo successful metamorphosis into adults is negatively influenced in the absence of SOD2. We therefore determined the impact of a high superoxide environment on cell size, progression through the cell cycle, cell differentiation, and cell death and found no difference between SOD2-null and SOD2+ larva and pupa. Thus loss of SOD2 activity clearly has no effect on pre-adult imaginal tissues. Instead, we found that the high mitochondrial superoxide environment arising from the absence of SOD2 leads to the induction of autophagy. Such autophagic response may underpin the resistance of pre-adult tissues to unscavenged ROS. Finally, while our data establish that SOD2 activity is less essential for normal development, the mortality of Sod2-/- neonates of both Drosophila and mice suggests that SOD2 activity is indeed essential for the viability of adults. We therefore asked if the early mortality of SOD2-null young adults could be rescued by activation of SOD2 expression. The results support the conclusion that the early mortality of SOD2-null adults is largely attributable to the absence of SOD2 activity in the adult per se. This finding somewhat contradicts the widely held notion that failure to scavenge the high volume of superoxide emanating from the oxidative demands of development would be highly detrimental to developing tissues.

Abstract 5019: HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice

Emerging evidence suggests that cancer cells undergo chromatin remodeling and epigenetic reprogramming to co-opt stem cell properties and drive tumor progression. The HMGA1 chromatin remodeling protein is an architectural transcription factor that binds to DNA at AT-rich sequences where it “opens” chromatin, recruits transcriptional complexes, and modulates gene expression. The HMGA1 gene is highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in differentiated tissues. HMGA1 becomes re-expressed in most high-grade cancers and high levels portend adverse clinical outcomes. In colon cancer, HMGA1 is among the genes most highly overexpressed compared to normal intestinal epithelium. We previously reported that HMGA1 drives tumor progression in colon cancer by inducing stem cell genes involved in an epithelial-mesenchymal transition. We also discovered that Hmga1 transgenic mice develop marked proliferative changes and pre-malignant polyposis in the intestinal epithelium. To determine how Hmga1 functions in the intestines during tissue homeostasis and carcinogenesis, we examined in transgenic mice and organoid models. Here, we uncover a novel role for Hmga1 in maintaining the intestinal stem cell (ISC) pool and Paneth cell niche. Hmga1 is required by ISCs to organize into three-dimensional organoids in vitro; silencing Hmga1 disrupts organoid formation and bud development. Conversely, overexpression of Hmga1 increases organoid formation, bud development, and replating efficiency, suggesting that Hmga1 enhances ISC function and/or number. We therefore crossed the Hmga1 transgenic mice onto the Lgr5-EGFP background to enumerate ISCs and found that Hmga1 expands the ISC compartment. To determine how this occurs, we performed in vivo imaging and discovered that Hmga1 enhances self-renewal of ISCs. Mechanistically, we found that Hmga1 amplifies Wnt/β-catenin signaling by inducing genes encoding both Wnt agonist receptors and downstream Wnt target genes. Surprisingly, Hmga1 also expands the Paneth cell niche, which is comprised of terminally differentiated crypt cells that secrete Wnt to support ISCs. Because Paneth cells require Sox9 for development, we determined whether Hmga1 regulates its expression. Hmga1 binds directly to the Sox9 promoter at 2 AT-rich sites to activate its expression. In human colonic epithelium, HMGA1 and SOX9 are positively correlated, and both become markedly up-regulated in colon carcinogenesis. This work not only provides new insights into the role of Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment, but also suggests that deregulated Hmga1 perturbs this equilibrium during polyposis and carcinogenesis. Our results also highlight the HMGA1-WNT-SOX9 pathway as rational therapeutic target in colon carcinogenesis. Citation Format: Lingling Xian, Dan Georgess, Li Luo, Lionel Chia, Qihua Gu, Tait Huso, Amy Belton, David Huso, Andrew Ewald, Linda M.S. Resar. HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice [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 5019. doi:10.1158/1538-7445.AM2017-5019

Abstract 3122: HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite advances in our ability to detect and treat breast cancer, it remains a leading cause of death in women with cancer worldwide, and the incidence is rising. Approximately 15-20% of all cases are classified as triple negative breast cancer (TNBC), a subtype that is frequently associated with rapid progression and poor outcome. TNBC refers to the lack of detectable markers for the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu amplification. These tumors do not respond to our most effective and least toxic therapies, including hormonal therapy (tamoxifen) or trastuzumab. We are studying molecular pathways that lead to tumor progression in TNBC and can be targeted with novel therapies. Our focus is the high mobility group A1 (HMGA1) oncogene. HMGA1 is highly expressed during embryogenesis, with low or undetectable levels in differentiated, adult tissues. HMGA1 is enriched in virtually all high-grade (poorly differentiated) cancers studied to date, including TNBCs, and high expression portends a poor prognosis in breast and other cancers. To investigate the role of HMGA1 in tumor progression in breast cancer, we silenced HMGA1 expression in TNBC cell lines (MDA-MB-231, Hs578T) using lentiviral-mediated delivery of short hairpin RNA. Strikingly, proliferation was markedly impaired, and many cells underwent apoptotic cell death within 5 days following HMGA1 knock-down. Surprisingly, cell morphology also changed dramatically, whereby the fibroblast-like, spindle-shaped cells became cuboidal and epithelial-like, consistent with a mesenchymal-epithelial transition, or MET. E-CADHERIN mRNA was induced, while both SNAIL and VIMENTIN were repressed in the knock-down cells, also consistent with MET. In addition, silencing HMGA1 blocked migration, invasion, and the formation of tumor foci in the lungs following tail vein injection of MDA-MB-231 cells. Moreover, both primary tumorigenesis and metastatic progression to the lungs were markedly inhibited in MDA-MB-231 cells with knock-down of HMGA1 following implantation in mammary fat pads. Furthermore, silencing HMGA1 blocked primary and secondary mammosphere formation, indicating that HMGA1 is required for this stem cell property. Tumorigenesis experiments at limiting dilutions showed that silencing HMGA1 depletes the tumor initiator/cancer stem cell pool. Using global gene expression analysis, we identified an HMGA1 signature of differentially-regulated genes in the control cells compared to knock-down cells. We also found that the HMGA1 signature is highly enriched in embryonic stem cells. Together, these findings indicate that silencing HMGA1 reprograms invasive, mesenchymal TNBCs into non-invasive, epithelial-like cells with slower growth and an altered gene expression signature. Studies are now needed to determine how to target HMGA1 in therapy. Citation Format: Sandeep N. Shah, Leslie Cope, Amy Belton, Weijie Poh, Saraswati Sukumar, David Huso, Linda Resar. HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3122. doi:10.1158/1538-7445.AM2013-3122

Abstract 429: The high mobility group A1 oncogene enhances cellular reprogramming to a pluripotent stem-like cell

The molecular mechanisms that enable cancer cells to metastasize are poorly understood, although emerging evidence indicates that transcriptional networks required for stem cell properties during embryogenesis are co-opted by cancer cells during metastatic progression. To elucidate the molecular underpinnings of “stemness” in cancer and normal development, we investigated transcriptional networks and epigenetic alterations during the induction of pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes proteins that bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. Our group first discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive tumors in transgenic mice. Recent studies also identified HMGA1 as a key transcription factor enriched in human embryonic stem (hESCs) cells, induced pluripotent stem cells (iPSCs), refractory leukemia, and high-grade/poorly differentiated cancers arising from diverse tissues. Together, these findings are consistent with the hypothesis that HMGA1 drives an undifferentiated, stem cell-like state during malignant transformation and normal development. To further investigate the role of HMGA1 in the stem cell state, we assessed its function in the derivation of iPSCs using multiple approaches. Here, we demonstrate for the first time that HMGA1 significantly enhances the reprogramming of somatic cells (bone marrow-derived mesenchymal stem cells, fetal lung cells, or mononuclear blood cells) into iPSCs together with OCT4, SOX2, KLF4, and cMYC (OSKM) using a retroviral or episomal, non-integrating approach. When hESCs are induced to differentiate, HMGA1 expression falls and parallels that of other pluripotency factors, such as OCT4, NANOG, and SOX2. We also found that forced expression of HMGA1 blocks differentiation of hESCs. To determine how HMGA1 induces a stem cell state, we assessed gene expression and epigenetic changes. During the reprogramming process, HMGA1 induces the expression of pluripotency and cancer genes, including SOX2, LIN28, and cMYC, while in hESCs, knock-down of HMGA1 results in repression of these genes. In addition, NANOG and OCT4 are repressed in hESCs following knock-down of HMGA1. By chromatin immunoprecipitation, HMGA1 binds to the promoters of SOX2, LIN28, and cMYC in vivo in hESCs. Moreover, HMGA1 is associated with decreased promoter methylation of select stem cell genes early in reprogramming. These findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks and epigenetic remodeling that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in poorly differentiated, stem-like cancers or exploited in regenerative medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 429. doi:1538-7445.AM2012-429

Abstract 3039: The high mobility group A1 oncogene & NF-κB cooperate in malignant transformation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Our research is directed at elucidating key transcriptional networks and underlying cellular pathways regulated by the high mobility group A1 (HMGA1) gene in refractory malignancies. Work from our group and others has shown that blocking HMGA1 interferes with multiple oncogenic phenotypes, including proliferation, anchorage-independent cell growth, migration, invasion, xenograft tumorigenicity, and metastatic progression in murine models. HMGA1 was also identified as a key transcription factor enriched in poorly differentiated solid tumors, leukemic stem cells, and embryonic stem (ES) cells. Importantly, previous studies on diverse tumors show that patients with tumors overexpressing HMGA1 have poor clinical outcomes. Together, these findings suggest that HMGA1 is a major regulator in diverse, poorly differentiated tumors and embryonic stem cells, although the molecular mechanisms through which HMGA1 functions remain unclear. We have begun to identify a few downstream genes and cellular pathways activated by HMGA1 in transformation. In gene expression profile analysis, we found that HMGA1 induces genes involved in mediating inflammatory signals. We identified NF-κB as a major node and inflammation as a major pathway. In previously published studies, we showed that HMGA1 induces expression of STAT3, COX-2, and MMP-2, genes which function in transformation and inflammation. Moreover, we found that anti-inflammatory agents block tumorigenesis in our HMGA1 transgenic mice and in xenograft tumors from aggressive human cancer cells overexpressing HMGA1. Together, these findings are consistent with the hypothesis that HMGA1 functions together with NF-κB to drive inflammatory pathways, induce transformation, and promote a poorly differentiated, stem-like state. To test this hypothesis and dissect the role of NF-κB in transformation induced by HMGA1, we have begun genetic experiments with HMGA1 transgenic mice and mice deficient in the major components of NF-κB. The NF-κB transcriptional complex is comprised primarily of p65:p50 heterodimers in which p65 functions as an activator, and p50, which lacks a transcriptional activation domain, is thought to function as an inhibitor. The p50/p65 heterodimers are thought to activate transcription of canonical NF-κB genes, whereas p50 homodimers repress transcription. Indeed, mice deficient in p50 have been shown to have enhanced NF-κB activity and inflammatory responses. In our preliminary studies, we found that the lymphoid tumors are significantly larger in the mice that are transgenic for HMGA1 and deficient in the inhibitory p50 subunit. This suggests that the excess in the p65 “activator” could lead to enhanced tumorigenesis induced by HMGA1. These studies should not only enhance our understanding of pathways associated with HMGA1 in malignant transformation, but also uncover novel biomarkers and therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3039. doi:1538-7445.AM2012-3039

Abstract 4243: HMGA1: A driver of the stem cell phenotype in intestinal cyrpt cells and colon cancer

Although the HMGA1 gene is highly expressed in human embryonic stem cells and high-grade, poorly differentiated cancers, its function in these settings has not been clearly elucidated. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which are members of a family of non-histone, chromatin remodeling proteins. These proteins bind to the minor groove of AT-rich regions in the B-form of DNA, which leads to the recruitment of transcriptional factors that modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis and in aggressive human cancers arising from different embryologic origins, including cancers of hematopoietic, lung, breast, prostate, and colon cells. Forced overexpression of HMGA1a or HMGA1b induces a transformed phenotype in cultured cells and inhibiting HMGA1 blocks transformation in human cancer cell lines. Because HMGA1 functions in transcriptional regulation, we hypothesize that it induces neoplastic transformation by dysregulating specific molecular pathways. To investigate the pathways disrupted by HMGA1, we are studying our HMGA1a transgenic mice as a model system. As previously reported, these mice develop aggressive T-cell leukemia with complete penetrance and females also develop uterine cancer. At necropsy, we also discovered polyps involving the small and large intestines. Histopathologically, the transgenic intestines are characterized by a thickened mucosal surface, hyperproliferative changes, and hamartomatous polyps. Immunohistochemical analysis reveals an increase in the population of intestinal cells expressing the colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr-5) in transgenic compared to wildtype mice. These data support our observation of an expansion in the crypt progenitor/intestinal stem cell populations. Our preliminary results also show that inhibiting HMGA1 expression in colon cancer cell lines interferes with anchorage-independent cell growth and tumorigenesis, indicating that HMGA1 is required for these transformation phenotypes in colon cancer. In addition, we found that HMGA1 is enriched in >70% of primary tumors with high-grade colon cancer. These findings suggest that HMGA1 is important in maintaining the stem cell niche in the intestine. Moreover, HMGA1 could promote neoplastic transformation and tumor progression in the intestine by driving a stem-like phenotype in colonic cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4243.