ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

In vivo loss of tumorigenicity in a patient-derived orthotopic xenograft mouse model of ependymoma.

Introduction: Ependymomas (EPN) are the third most common malignant brain cancer in children. Treatment strategies for pediatric EPN have remained unchanged over recent decades, with 10-year survival rates stagnating at just 67% for children aged 0-14 years. Moreover, a proportion of patients who survive treatment often suffer long-term neurological side effects as a result of therapy. It is evident that there is a need for safer, more effective treatments for pediatric EPN patients. There are ten distinct subgroups of EPN, each with their own molecular and prognostic features. To identify and facilitate the testing of new treatments for EPN, in vivo laboratory models representative of the diverse molecular subtypes are required. Here, we describe the establishment of a patient-derived orthotopic xenograft (PDOX) model of posterior fossa A (PFA) EPN, derived from a metastatic cranial lesion. Methods: Patient and PDOX tumors were analyzed using immunohistochemistry, DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. Results: Both patient and PDOX tumors classified as PFA EPN by methylation profiling, and shared similar histological features consistent with this molecular subgroup. RNA sequencing revealed that gene expression patterns were maintained across the primary and metastatic tumors, as well as the PDOX. Copy number profiling revealed gains of chromosomes 7, 8 and 19, and loss of chromosomes 2q and 6q in the PDOX and matched patient tumor. No clinically significant single nucleotide variants were identified, consistent with the low mutation rates observed in PFA EPN. Overexpression of EZHIP RNA and protein, a common feature of PFA EPN, was also observed. Despite the aggressive nature of the tumor in the patient, this PDOX was unable to be maintained past two passages in vivo. Discussion: Others who have successfully developed PDOX models report some of the lowest success rates for EPN compared to other pediatric brain cancer types attempted, with loss of tumorigenicity not uncommon, highlighting the challenges of propagating these tumors in the laboratory. Here, we discuss our collective experiences with PFA EPN PDOX model generation and propose potential approaches to improve future success in establishing preclinical EPN models.

Conventional Therapies Deplete Brain-Infiltrating Adaptive Immune Cells in a Mouse Model of Group 3 Medulloblastoma Implicating Myeloid Cells as Favorable Immunotherapy Targets.

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments.

Defining the molecular features of radiation-induced glioma: A systematic review and meta-analysis.

BACKGROUND: Cranial radiation therapy is essential in treating many pediatric cancers, especially brain tumors; however, its use comes with the risk of developing second malignancies. Cranial radiation-induced gliomas (RIGs) are aggressive high-grade tumors with a dismal prognosis, for which no standard therapy exists. A definitive molecular signature for RIGs has not yet been established. We sought to address this gap by performing a systematic review and meta-analysis of the molecular features of cranial RIGs. METHODS: A systematic review of the literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles and case reports that described molecular analyses of cranial radiation-induced high-grade gliomas were identified and evaluated, and data extracted for collation. RESULTS: Of 1727 records identified, 31 were eligible, containing 102 unique RIGs with molecular data. The most frequent genetic alterations in RIGs included PDGFRA or TP53 mutations, PDGFRA or CDK4 amplifications, and CDKN2A deletion, along with 1q gain, 1p loss and 13q loss. Of note, mutations in ACVR1, EGFR, H3F3A, HIST1H3B, HIST1H3C, IDH2, SMARCB1 or the TERT promoter were not observed. A comparative analysis revealed that RIGs are molecularly distinct from most other astrocytomas and gliomas and instead align most closely with the pedGBM_RTK1 subgroup of pediatric glioblastoma. CONCLUSIONS: This comprehensive analysis highlights the major molecular features of RIGs, demonstrates their molecular distinction from many other astrocytomas and gliomas, and reveals potential genetic drivers and therapeutic targets for this currently fatal disease.

Veliparib Is an Effective Radiosensitizing Agent in a Preclinical Model of Medulloblastoma.

Medulloblastoma is the most common malignant childhood brain tumor, and 5-year overall survival rates are as low as 40% depending on molecular subtype, with new therapies critically important. As radiotherapy and chemotherapy act through the induction of DNA damage, the sensitization of cancer cells through the inhibition of DNA damage repair pathways is a potential therapeutic strategy. The poly-(ADP-ribose) polymerase (PARP) inhibitor veliparib was assessed for its ability to augment the cellular response to radiation-induced DNA damage in human medulloblastoma cells. DNA repair following irradiation was assessed using the alkaline comet assay, with veliparib inhibiting the rate of DNA repair. Veliparib treatment also increased the number of γH2AX foci in cells treated with radiation, and analysis of downstream pathways indicated persistent activation of the DNA damage response pathway. Clonogenicity assays demonstrated that veliparib effectively inhibited the colony-forming capacity of medulloblastoma cells, both as a single agent and in combination with irradiation. These data were then validated in vivo using an orthotopic implant model of medulloblastoma. Mice harboring intracranial D425 medulloblastoma xenografts were treated with vehicle, veliparib, 18 Gy multifractionated craniospinal irradiation (CSI), or veliparib combined with 18 Gy CSI. Animals treated with combination therapy exhibited reduced tumor growth rates concomitant with increased intra-tumoral apoptosis observed by immunohistochemistry. Kaplan-Meier analyses revealed a statistically significant increase in survival with combination therapy compared to CSI alone. In summary, PARP inhibition enhanced radiation-induced cytotoxicity of medulloblastoma cells; thus, veliparib or other brain-penetrant PARP inhibitors are potential radiosensitizing agents for the treatment of medulloblastoma.

Small-molecule screen reveals synergy of cell cycle checkpoint kinase inhibitors with DNA-damaging chemotherapies in medulloblastoma.

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely, MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that cell cycle checkpoint kinase (CHK1/2) inhibition synergistically enhanced the cytotoxic activity of clinically used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.

A Novel Orthotopic Patient-Derived Xenograft Model of Radiation-Induced Glioma Following Medulloblastoma.

Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy. We report a case of RIG that arose in the brain stem following treatment for paediatric medulloblastoma, and the development and characterisation of a matched orthotopic patient-derived xenograft (PDX) model (TK-RIG915). Patient and PDX tumours were analysed using DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. While initially thought to be a diffuse intrinsic pontine glioma (DIPG) based on disease location, results from methylation profiling and WGS were not consistent with this diagnosis. Furthermore, clustering analyses based on RNA expression suggested the tumours were distinct from primary DIPG. Additional gene expression analysis demonstrated concordance with a published RIG expression profile. Multiple genetic alterations that enhance PI3K/AKT and Ras/Raf/MEK/ERK signalling were discovered in TK-RIG915 including an activating mutation in PIK3CA, upregulation of PDGFRA and AKT2, inactivating mutations in NF1, and a gain-of-function mutation in PTPN11. Additionally, deletion of CDKN2A/B, increased IDH1 expression, and decreased ARID1A expression were observed. Detection of phosphorylated S6, 4EBP1 and ERK via immunohistochemistry confirmed PI3K pathway and ERK activation. Here, we report one of the first PDX models for RIG, which recapitulates the patient disease and is molecularly distinct from primary brain stem glioma. Genetic interrogation of this model has enabled the identification of potential therapeutic vulnerabilities in this currently incurable disease.

New therapeutic opportunities from dissecting the pre-B leukemia bone marrow microenvironment.

The microenvironments of leukemia and cancer are critical for multiple stages of malignancies, and they are an attractive therapeutic target. While skeletal abnormalities are commonly seen in children with acute lymphoblastic leukemia (ALL) prior to initiating osteotoxic therapy, little is known about the alterations to the bone marrow microenvironment during leukemogenesis. Therefore, in this study, we focused on the development of precursor-B cell ALL (pre-B ALL) in an immunocompetent BCR-ABL1+ model. Here we show that hematopoiesis was perturbed, B lymphopoiesis was impaired, collagen production was reduced, and the number of osteoblastic cells was decreased in the bone marrow microenvironment. As previously found in children with ALL, the leukemia-bearing mice exhibited severe bone loss during leukemogenesis. Leukemia cells produced high levels of receptor activator of nuclear factor κB ligand (RANKL), sufficient to cause osteoclast-mediated bone resorption. In vivo administration of zoledronic acid rescued leukemia-induced bone loss, reduced disease burden and prolonged survival in leukemia-bearing mice. Taken together, we provide evidence that targeting leukemia-induced bone loss is a therapeutic strategy for pre-B ALL.

Deregulated expression of connective tissue growth factor (CTGF/CCN2) is linked to poor outcome in human cancer.

Connective tissue growth factor (CTGF/CCN2) has long been associated with human cancers. The role it plays in these neoplasms is diverse and tumour specific. Recurring patterns in clinical outcome, histological desmoplasia and mechanisms of action have been found. When CTGF is overexpressed compared to low-expressing normal tissue or is underexpressed compared to high-expressing normal tissue, the functional outcome favours tumour survival and disease progression. CTGF acts by altering proliferation, drug resistance, angiogenesis, adhesion and migration contributing to metastasis. The pattern of CTGF expression and tumour response helps to clarify the role of this matricellular protein across a multitude of human cancers.

Inhibition of the JAK2/STAT3 pathway reduces gastric cancer growth in vitro and in vivo.

Signal Transducer and Activator of Transcription-3 (STAT3) is constitutively activated in many cancers where it promotes growth, inflammation, angiogenesis and inhibits apoptosis. We have shown that STAT3 is constitutively activated in human gastric cancer, and that chronic IL-11-driven STAT3 transcriptional activity induces gastric tumourigenesis in the gp130(757FF) mouse model of gastric cancer development. Here we show that treatment of human AGS gastric cancer cells with the Janus Kinase (JAK) inhibitor WP1066 dose-, and time-dependently inhibits STAT3 phosphorylation, in conjunction with reduced JAK2 phosphorylation, reduced proliferation and increased apoptosis. In addition, application of intraperitoneal WP1066 for 2 weeks, reduced gastric tumour volume by 50% in the gp130(757FF) mouse coincident with reduced JAK2 and STAT3 activation compared with vehicle-treated, littermate controls. Gastric tumours from WP1066- treated mice had reduced polymorphonuclear inflammation, coincident with inhibition of numerous proinflammatory cytokines including IL-11, IL-6 and IL-1ß, as well as the growth factors Reg1 and amphiregulin. These results show that WP1066 can block proliferation, reduce inflammation and induce apoptosis in gastric tumour cells by inhibiting STAT3 phosphorylation, and that many cytokines and growth factors that promote gastric tumour growth are regulated by STAT3-dependent mechanisms. WP1066 may form the basis for future therapeutics against gastric cancer.

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis.

IL-11 is a parietal cell cytokine that induces atrophic gastritis.

BACKGROUND AND AIMS: IL-is important in gastric damage, mucosal repair and gastric cancer progression. We analysed IL-11 expression in H.pylori infected mouse stomach, the site of gastric IL-11 expression in mice and humans, and the effect of exogenous IL-11 on gastric mucosal homeostasis. METHODS: IL-11 protein was localised in mouse and human stomach. The impact of chronic, exogenous IL-11 on normal mouse stomach was examined histologically and transcriptionally by microarray, confirmed by mRNA and protein analysis. Functional impact of IL-11 on gastric acid secretion was determined. RESULTS: In mice infected with H.pylori, IL-11 was increased in fundic mucosa with temporal expression similar to IL-1b. IL-11 protein was localised predominantly to parietal cells in mouse and human stomach. Application of exogenous IL-11 to resulted in fundic parietal and chief cell loss, hyperplasia, mucous cell metaplasia and inflammation. Coincident with cellular changes were an increased gastric pH, altered parietal cell ultrastructure and altered gene expression, particularly genes involved in immune response and ion transport which could result in compromised acid secretion. We confirmed that a single dose of IL-11 effectively ablated the gastric response to histamine. CONCLUSIONS: IL-11 is a parietal cell cytokine that blocks gastric acid secretion, likely via reducing expression of parietal cell ion transport genes, CCKb and histamine H2 receptors. IL-11 expression is increased in H. pylori infected mouse stomach and treatment of wild type mice with IL-11 induced changes in the gastric fundic mucosa reminiscent of chronic atrophic gastritis, a precursor to gastric cancer.

The interleukin-6 family cytokine interleukin-11 regulates homeostatic epithelial cell turnover and promotes gastric tumor development.

BACKGROUND & AIMS: Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. METHODS: We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. RESULTS: IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor alpha significantly reduced HKbeta-/- mouse fundic hyperplasia and ablated gp130(757F/F) mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-beta, RegIII-gamma, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130(757F/F) and HKbeta-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. CONCLUSIONS: Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.

Cytokine signalling by gp130 regulates gastric mucosal healing after ulceration and, indirectly, antral tumour progression.

The cytokines IL-6 and IL-11, which signal via the receptor gp130, have been implicated in various gut pathologies, including inflammation and wound healing. We used mouse cytokine signalling mutants to evaluate the role of gp130 pathways in gastric ulceration and healing and the effect of spatially remote fundic ulceration on antral tumour progression, since compromised wound healing may impact tumourigenesis. Glacial acetic acid applied to the serosal surface of stomachs from wild-type, gp130(757FF), IL-6(-/-) and IL-11 receptor (R)alpha(-/-) mice was used to induce discrete haemostasis/necrosis and resultant mucosal ulceration. Wound pathology and mRNA expression of key cytokine target genes were examined 2 and 14 weeks after ulcer induction. The outcome of fundic ulceration on antral tumour development in gp130(757FF) mice was also examined. Chemical haemostasis in gp130(7575FF) mice produces more severe gastric ulcers than in wild-type mice. Lack of IL-6 produces more severe ulceration, while loss of IL-11Ralpha less severe ulcers, suggesting a role for IL-11 in ulcer induction. Increased expression of ulcer-associated IL-11 and its established mitogenic target genes RegI, IIIbeta and IIIgamma paralleled severe ulceration in gp130(757FF) mice. In this model, coincident with fundic ulceration, antral tumour development was inhibited and correlated with decreased RegI, IIIbeta and IIIgamma and reduced MMP9 and 13 expression. IL-11-driven transcription via gp130 contributes to the gastric mucosal response to ulceration. Fundic mucosal ulceration modulates antral growth factor and metalloproteinase gene expression, thereby contributing to restricted tumour growth.

Differential regulation of gastric tumor growth by cytokines that signal exclusively through the coreceptor gp130.

BACKGROUND & AIMS: We have shown that mice with a mutation in gp130 (gp130(757F/F)), the signal transducing receptor for interleukin (IL)-6 family cytokines, have chronic gastric inflammation and develop distal stomach tumors associated with deregulated phosphorylated STAT3 expression. This model recapitulates many characteristics of intestinal-type gastric cancer in humans. METHODS: To evaluate the role of IL-6 and IL-11 as ligands regulating tumor growth and submucosal invasion, we compared tumor characteristics of gp130(757F/F) mice with gp130(757F/F) mice lacking IL-6 or mature T and B cells. RESULTS: As a result of the gp130(757F/F) mutation, expression of IL-6 and IL-11 was greatly up-regulated concomitant with activation of STAT3 and development of tumors. However, the lack of IL-6 or T and B cells did not impact on tumor growth. While IL-6 did not regulate tumor growth or tumor vascularization, gp130(757F/F)/IL-6(-/-) mice showed approximately 10-20-fold more submucosal tumor invasion, reduced mononuclear inflammatory cell infiltrate, and greater IL-11 and matrix metalloproteinase (MMP)-13 and MMP-9 synthesis than gp130(757F/F) mice. Expression of MMP-13 was largely restricted to tumor-associated stroma, but MMP-9 was also expressed in polymorphonuclear cells and a subset of epithelial cells. In addition, treatment with recombinant IL-11 stimulated expression of MMP-13 and MMP-9 in stomachs of wild-type mice. CONCLUSIONS: Increased submucosal invasion in gp130(757F/F)/IL-6(-/-) mice could not be explained by increased vascularization or reduced immunosurveillance but was most likely facilitated by augmented metalloproteinase activity driven by elevated IL-11 levels.

Cross-fostering in Macropus eugenii leads to increased weight but not accelerated gastrointestinal maturation.

Stomach and small intestine development was characterized in tammar wallaby (Macropus eugenii) pouch young (PY) using both morphological and immunohistological criteria. At birth, the stomach is undeveloped in comparison to the well-developed intestinal mucosa. The stomach maintains a uniform morphology in both the forestomach and hindstomach regions until the specialization of cardiac and gastric glands are seen at PY170. Parietal cells, found throughout the mucosa are downregulated in the forestomach as cardiac glandular stomach is developing prior to the transition of the offspring to a diet that includes herbage. In the small intestine, mature-type villi are present at birth but the muscularis externa is immature and undergoes significant development around PY120 onwards. We investigated the effects of changes in maternal milk on gut development in the tammar wallaby using a cross fostering approach that provided younger pouch young with older stage milk. Younger PY (average age 67 days postpartum, n = 5) were transferred onto teats vacated by older stage PY (average age 100 days postpartum, n = 6) for 34 days before gut development was assessed. In addition milk analysis was performed before and after fostering events. Cross-fostered PY animals receiving older stage milk were found to be 31% heavier than controls. There was no difference between carbohydrate and protein concentrations however, fostered PY milk had a higher concentration of lipid than that of controls that may have contributed to heavier fostered PY. No difference was found in stomach or small intestine development between these groups using the criteria employed in this study.

Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co-receptor gp130.

BACKGROUND AND AIMS: We have developed a mouse model of gastric cancer that resembles human intestinal-type adenocarcinoma. The aim of this study was to determine the identity and temporal changes in mediators of IL-6 signaling regulating tumor development. METHODS: gp130(757F/F) Mice that lack the SHP2-binding site on the IL-6 family receptor gp130 and have increased STAT 3 activity and wild-type littermates were used. Cohorts were assessed by quantitative histology and immunohistochemistry for gastric cell phenotype and proliferation markers from 4 to 40 weeks of tumor development. Northern blotting and in situ hybridization were used to quantify expression of the tumor suppressor TFF1 and the mitogens gastrin and Reg I. Expression of epidermal growth factor receptor (EGFr) and its ligands was measured by RT-PCR analysis. Age-matched differences in gene expression profiles were tested by ANOVA. RESULTS: Hyperplastic antral tumors with inflammation and ulceration were evident in gp130(757F/F) mice at 4 weeks of age and reached maximum size by 20 weeks. Tumor progression was marked by gastritis, atrophy, intestinal metaplasia, dysplasia, and submucosal invasion after 30 weeks. Both TFF1 and gastrin expression were progressively inhibited during tumorigenesis, whereas Reg I was stimulated. The EGFr and its ligands transforming growth factor (TGF)-alpha and heparin-binding EGF had increased expression corresponding to maximal tumor growth. CONCLUSIONS: gp130(757F/F) Mice rapidly develop distal stomach tumors, with loss of SHP2/Erk/AP-1 transcriptional regulation exemplified by decreased TFF1 expression and increased STAT1/3 regulated genes such as Reg I. Tumor development occurs in a hypogastrinemic environment. Balanced IL-6 signaling is required for maintaining gastric homeostasis.

Insights into the mechanisms of gastric adaptation to aspirin-induced injury: a role for regenerating protein but not trefoil peptides.

The phenomenon of reduced gastric mucosal injury despite repeated doses of a damaging agent is termed adaptation. Adaptation to nonsteroidal anti-inflammatory drug-induced injury has been clearly demonstrated in both humans and experimental animals; however, the precise mechanisms remain unclear. We hypothesized that mediators of adaptation might be the regenerating protein (RegI) and the trefoil peptides TFF1 and TFF2, because these proteins play pivotal roles in gastric mucosal protection and repair. The gene expression and the protein levels of these proteins were measured and compared in normal, aspirin-injured, and aspirin-adapted rat stomachs. TFF gene and protein expression levels were similar in all three groups, whereas RegI gene expression and protein levels in adapted stomach were increased. A time course analysis of RegI expression during the onset and offset of adaptation showed that mucosal RegI increased during the development of adaptation, was maintained during subsequent aspirin dosing, and returned to baseline levels once dosing had ceased and adaptation was lost-indicative of a causal role in the adaptation process. Colocalization of increased RegI with gastric epithelial areas showing increased proliferation also suggests that RegI may be an important mediator of the resolution of mucosal injury that is characteristic of gastric adaptation to aspirin.