Loss of AMPKα2 Impairs Hedgehog-Driven Medulloblastoma Tumorigenesis

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that has a dual role in cancer, i.e., pro- or anti-tumorigenic, depending on the context. In medulloblastoma, the most frequent malignant pediatric brain tumor, several in vitro studies previously showed that AMPK suppresses tumor cell growth. The role of AMPK in this disease context remains to be tested in vivo. Here, we investigate loss of AMPKα2 in a genetically engineered mouse model of sonic hedgehog (SHH)-medulloblastoma. In contrast to previous reports, our study reveals that AMPKα2 KO impairs SHH medulloblastoma tumorigenesis. Moreover, we performed complementary molecular and genomic analyses that support the hypothesis of a pro-tumorigenic SHH/AMPK/CNBP axis in medulloblastoma. In conclusion, our observations further underline the context-dependent role of AMPK in cancer, and caution is warranted for the previously proposed hypothesis that AMPK agonists may have therapeutic benefits in medulloblastoma patients. Note: an abstract describing the project was previously submitted to the American Society for Investigative Pathology PISA 2018 conference and appears in The American Journal of Pathology (Volume 188, Issue 10, October 2018, Page 2433).

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Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.352-361.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 352-361 ◽

Cited By ~ 32

Author(s):

Heather A. Wiatrowski ◽

Bryce J. W. van Denderen ◽

Cristin D. Berkey ◽

Bruce E. Kemp ◽

David Stapleton ◽

...

Keyword(s):

Glycogen Synthase ◽

Metabolic Stress ◽

Binding Domain ◽

Glycogen Accumulation ◽

Snf1 Kinase ◽

Transcriptional Regulatory ◽

Amp Activated Protein Kinase

ABSTRACT The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK β1 subunit, and the sequence is conserved in the Snf1 kinase β subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK β1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

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CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms

Endocrine Related Cancer ◽

10.1530/erc-14-0152 ◽

2014 ◽

Vol 21 (6) ◽

pp. 879-890 ◽

Cited By ~ 6

Author(s):

Sebastian Krug ◽

Benjamin Kühnemuth ◽

Heidi Griesmann ◽

Albrecht Neesse ◽

Leonie Mühlberg ◽

...

Keyword(s):

Tumour Progression ◽

Treatment Options ◽

Genetically Engineered ◽

Neuroendocrine Neoplasms ◽

Dependent Manner ◽

Rare Tumour ◽

Pancreatic Neuroendocrine Neoplasms ◽

Genetically Engineered Mouse Model

Pancreatic neuroendocrine neoplasms (PNENs) constitute a rare tumour entity, and prognosis and treatment options depend on tumour-mediating hallmarks such as angiogenesis, proliferation rate and resistance to apoptosis. The molecular pathways that determine the malignant phenotype are still insufficiently understood and this has limited the use of effective combination therapies in the past. In this study, we aimed to characterise the effect of the oncogenic transcription factor Cut homeobox 1 (CUX1) on proliferation, resistance to apoptosis and angiogenesis in murine and human PNENs. The expression and function ofCUX1were analysed using knockdown and overexpression strategies in Ins-1 and Bon-1 cells, xenograft models and a genetically engineered mouse model of insulinoma (RIP1Tag2). Regulation of angiogenesis was assessed using RNA profiling and functional tube-formation assays in HMEC-1 cells. Finally,CUX1expression was assessed in a tissue microarray of 59 human insulinomas and correlated with clinicopathological data.CUX1expression was upregulated during tumour progression in a time- and stage-dependent manner in the RIP1Tag2 model, and associated with pro-invasive and metastatic features of human insulinomas. Endogenous and recombinantCUX1expression increased tumour cell proliferation, tumour growth, resistance to apoptosis, and angiogenesisin vitroandin vivo. Mechanistically, the pro-angiogenic effect ofCUX1was mediated via upregulation of effectors such as HIF1α and MMP9.CUX1mediates an invasive pro-angiogenic phenotype and is associated with malignant behaviour in human insulinomas.

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In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers

Cancers ◽

10.3390/cancers12051150 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1150 ◽

Cited By ~ 3

Author(s):

Vincenza De Gregorio ◽

Francesco Urciuolo ◽

Paolo Antonio Netti ◽

Giorgia Imparato

Keyword(s):

Human Papillomavirus ◽

Tumor Microenvironment ◽

Critical Role ◽

Genetically Engineered ◽

Cancer Disease ◽

Raft Culture ◽

Squamous Epithelia

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

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Inhibition of Filamentation Can Be Used To Treat Disseminated Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00628-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3312-3316 ◽

Cited By ~ 89

Author(s):

Stephen P. Saville ◽

Anna L. Lazzell ◽

Alexander P. Bryant ◽

Angelika Fretzen ◽

Alex Monreal ◽

...

Keyword(s):

Antifungal Agents ◽

Developmental Process ◽

Mortality Rates ◽

Genetically Engineered ◽

In Vivo Studies ◽

Yeast Form ◽

Active Disease

ABSTRACT Candida albicans remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in C. albicans pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a C. albicans tet-NRG1 strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.

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A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase α1 in liver and lung

Biochemical Journal ◽

10.1042/bj20050269 ◽

2005 ◽

Vol 392 (1) ◽

pp. 201-209 ◽

Cited By ~ 20

Author(s):

Russell M. Crawford ◽

Kate J. Treharne ◽

O. Giles Best ◽

Richmond Muimo ◽

Claudia E. Riemen ◽

...

Keyword(s):

Protein Kinase ◽

Mammalian Cells ◽

Nucleoside Diphosphate Kinase ◽

Cell Types ◽

Acid Synthesis ◽

Cellular Fatty Acid ◽

Energy Status ◽

Amp Activated Protein Kinase

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (∼16–20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK α1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the α1 (but not α2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK α1-mediated phosphorylation is present in mammalian cells.

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Irisin alleviates obesity-related spermatogenesis dysfunction via the regulation of the AMPKα signalling pathway

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00821-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Yang Mu ◽

Huang-Guan Dai ◽

Ling-Bo Luo ◽

Jing Yang

Keyword(s):

Oxidative Stress ◽

Sperm Count ◽

Underlying Mechanism ◽

Signalling Pathway ◽

Protective Effects ◽

Exercise Induced ◽

Amp Activated Protein Kinase

Abstract Background Infertility is a common complication in obese men. Oxidative stress and testicular apoptosis play critical roles in obesity-induced spermatogenesis dysfunction. It has been reported that irisin, an exercise-induced myokine, may attenuate oxidative damage and testicular apoptosis in several diseases; however, its role in obesity-induced spermatogenesis dysfunction remains unclear. The purpose of this study was to investigate the role and underlying mechanism of irisin in obesity-induced dysfunction of spermatogenesis. Methods Male mice were fed a high-fat diet (HFD) for 24 weeks to establish a model of obesity-induced spermatogenesis dysfunction. To explore the effects of irisin, mice were subcutaneously infused with recombinant irisin for 8 weeks beginning at 16 weeks after starting a HFD. To confirm the role of AMP-activated protein kinase α (AMPKα), AMPKα-deficient mice were used. Results The data showed decreased serum irisin levels in obese patients, which was negatively correlated with sperm count and progressive motility. Irisin was downregulated in the plasma and testes of obese mice. Supplementation with irisin protected against HFD-induced spermatogenesis dysfunction and increased testosterone levels in mice. HFD-induced oxidative stress, endoplasmic reticulum (ER) stress and testicular apoptosis were largely attenuated by irisin treatment. Mechanistically, we identified that irisin activated the AMPKα signalling pathway. With AMPKα depletion, we found that the protective effects of irisin on spermatogenesis dysfunction were abolished in vivo and in vitro. Conclusions In conclusion, we found that irisin alleviated obesity-related spermatogenesis dysfunction via activation of the AMPKα signalling pathway. Based on these findings, we hypothesized that irisin is a potential therapeutic agent against obesity-related spermatogenesis dysfunction.

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Cyclocreatine suppresses prostate tumorigenesis through dual effects on SAM and creatine metabolism

10.1101/2021.02.26.432994 ◽

2021 ◽

Author(s):

Rachana Patel ◽

Lisa Rodgers ◽

Catriona A. Ford ◽

Linda K Rushworth ◽

Janis Fleming ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Progression ◽

De Novo ◽

Basal Respiration ◽

Genetically Engineered ◽

Prostate Tumorigenesis ◽

Genetically Engineered Mouse Model ◽

Creatine Metabolism

ABSTRACTProstate cancer is highly prevalent, being the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of PTEN and SPRY2 (Sprouty 2) tumour suppressors, we identified enhanced creatine metabolism within the phosphagen system in progressive disease. Altered creatine metabolism was validated in in vitro and in vivo prostate cancer models and in clinical cases. Upregulated creatine levels were due to increased uptake through the SLC6A8 creatine transporter and de novo synthesis, resulting in enhanced cellular basal respiration. Treatment with cyclocreatine (a creatine analogue that potently and specifically blocks the phosphagen system) dramatically reduces creatine and phosphocreatine levels. Blockade of creatine biosynthesis by cyclocreatine leads to cellular accumulation of S-adenosyl methionine (SAM), an intermediary of creatine biosynthesis, and suppresses prostate cancer growth in vitro. Furthermore, cyclocreatine treatment impairs cancer progression in our GEMM and in a xenograft liver metastasis model. Hence, by targeting the phosphagen system, cyclocreatine results in anti-tumourigenic effects from both SAM accumulation and suppressed phosphagen system.

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Disrupting Ectopic Super-Enhancers to Treat Multiple Myeloma

Blood ◽

10.1182/blood-2021-147551 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1593-1593

Author(s):

Seth Welsh ◽

Daniel Riggs ◽

Erin Meermeier ◽

Chang-Xin Shi ◽

Victoria Garbitt ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Mouse Model ◽

Therapeutic Response ◽

Genetically Engineered ◽

Therapeutic Window ◽

Super Enhancer ◽

Genetically Engineered Mouse Model

Abstract Multiple myeloma (MM) is an incurable form of plasma cell cancer in which primary and secondary chromosomal translocations routinely juxtapose oncogenes to plasma cell-specific super-enhancers. Coincidentally, drugs which target super-enhancers have had success clinically. For example, immunomodulatory imide drugs (IMiDs) degrade super-enhancer-binding pioneer factors IKAROS and AIOLOS, while glucocorticoids (Dexamethasone) and proteasome inhibitors (Bortezomib) have the ability to transrepress or block the processing of super-enhancer-forming NF-κB proteins, respectively. Currently, alternative enhancer-targeting drugs are also in clinical development, like p300 inhibitors which target the acetyl-binding bromodomains and/or histone acetyl transferase activity of the chromatin-regulating coactivator homologs CBP and EP300. Despite showing therapeutic promise, our understanding of how these drugs function, alone or together, remains incomplete. Case in point, we find that IMiD-induced degradation of its target proteins IKAROS and AIOLOS does not guarantee a therapeutic response in vitro, and patients successfully treated with IMiDs eventually relapse; meanwhile, coactivator-targeting therapies like p300 inhibitors are often too toxic in vivo, and lack a therapeutic window. To improve the outcomes of MM patients we need to understand the heterogeneous genetics and transcription-factor milieus of the myeloma enhancer landscape, as well as how to increase the precision of enhancer-disrupting drugs. To accomplish this, our lab utilizes more than 60 human myeloma cell lines that have been extensively characterized at the genetic, proteomic, and drug-therapeutic-response levels. Additionally, we have generated a highly-predictive immunocompetent mouse model (Vk*MYC hCRBN+) that develops human-like MM and is sensitive to both IMiDs and a new class of therapeutics termed "degronimids" (normal mice do not respond to IMiDs or degronimids). Our central hypothesis is that combining a broad coactivator-targeting drug (e.g., the p300 inhibitor GNE-781), with a MM-specific transcription factor-targeting drug (e.g., IMiDs) restricts toxicities to myeloma cells and thus improves the therapeutic window. Currently, we are testing a variety of coactivator-targeting compounds alongside traditional IMiD therapies and other preclinical transcription factor-targeting drugs both in vivo and in vitro. We show that Vk*MYC hCRBN+ mice are exquisitely sensitive to GNE-781, requiring one fourth of the dose needed to treat other cancers and therefore avoiding the neutropenia and thrombocytopenia seen at higher doses. Second, we show that although IMiDs and GNE-781 induce an effective but transient response in vivo as single agents, the combination of the two drugs proved curative, with a progressive deepening of the anti-tumor response occurring even after therapy is discontinued. Ongoing experiments aim to determine how this drug combination, and other coactivator + transcription factor-targeting combinations, permanently disrupt myeloma-specific super-enhancers. Disclosures Neri: BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy. Boise: AstraZeneca: Honoraria, Research Funding; AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees. Chesi: Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma.

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CRISP3 expression drives prostate cancer invasion and progression

Endocrine Related Cancer ◽

10.1530/erc-20-0092 ◽

2020 ◽

Vol 27 (7) ◽

pp. 415-430

Author(s):

Marianna Volpert ◽

Luc Furic ◽

Jinghua Hu ◽

Anne E O’Connor ◽

Richard J Rebello ◽

...

Keyword(s):

Prostate Cancer ◽

Secretory Protein ◽

Genetically Engineered ◽

Migration And Invasion ◽

Healthy Human ◽

Mouse Prostate ◽

Genetically Engineered Mouse Model ◽

Prostatic Epithelium

Identifying the factors stimulating prostate cancer cells migration and invasion has the potential to bring new therapeutic targets to the clinic. Cysteine-rich secretory protein 3 (CRISP3) is one of the most highly upregulated proteins during the transition of a healthy human prostatic epithelium to prostate cancer. Here we show using a genetically engineered mouse model of prostate cancer that CRISP3 production greatly facilitates disease progression from carcinoma in situ to invasive prostate cancer in vivo. This interpretation was confirmed using both human and mouse prostate cancer cell lines, which showed that exposure to CRISP3 enhanced cell motility and invasion. Further, using mass spectrometry, we show that CRISP3 induces changes in abundance of a subset of cell-cell adhesion proteins, including LASP1 and TJP1 both in vivo and in vitro. Collectively, these data identify CRISP3 as being pro-tumorigenic in the prostate and validate it as a potential target for therapeutic intervention.

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The H3K36me2 writer-reader dependency in H3K27M-DIPG

10.1101/2021.01.06.425580 ◽

2021 ◽

Author(s):

Jia-Ray Yu ◽

Gary LeRoy ◽

Devin Bready ◽

Joshua D. Frenster ◽

Ricardo Saldaña-Meyer ◽

...

Keyword(s):

Cellular Proliferation ◽

Histone H3 ◽

Pediatric Brain Tumor ◽

Diffuse Intrinsic Pontine Glioma ◽

Therapeutic Interventions ◽

Functional Pathway ◽

Proliferation In Vitro

AbstractThe lysine-to-methionine mutation at residue 27 of histone H3 (H3K27M) is a driving mutation in Diffuse Intrinsic Pontine Glioma (DIPG), a highly aggressive form of pediatric brain tumor with no effective treatment and little chance of survival. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity, the placement of methylation at lysine 27 of histone H3 (H3K27me2/3), promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation in vitro and tumorigenesis in vivo, and disrupts tumor-promoting gene expression programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers that mediate the pro-tumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Together, our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG and suggest a possibility to target this pathway for therapeutic interventions.

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