Loss of copy of MIR1-2 increases CDK4 expression in ileal neuroendocrine tumors.

Ileal neuroendocrine tumors (I-NETs) are the most common tumors of the small intestine. Although I-NETs are known for a lack of recurrently mutated genes, a majority of tumors do show loss of one copy of chromosome 18. Among the genes on chromosome 18 is MIR1-2, which encodes a microRNA, MIR1-3p, with high complementarity to the mRNA of CDK4. Here we show that transfection of neuroendocrine cell lines with MIR1-3p lowered CDK4 expression and activity, and arrested growth at the G1 stage of the cell cycle. Loss of copy of MIR1-2 in ileal neuroendocrine tumors associated with increased expression of CDK4. Genetic events that attenuated RB activity, including loss of copy of MIR1-2 as well as loss of copy of CDKN1B and CDKN2A, were more frequent in tumors from patients with metastatic I-NETs. These data suggest that inhibitors of CDK4/CDK6 may benefit patients whose I-NETs show loss of copy of MIR1-2, particularly patients with metastatic disease.

IGF2 drives formation of ileal neuroendocrine tumors in patients and mice.

By the strictest of definitions, a genetic driver of tumorigenesis should fulfill two criteria: it should be altered in a high percentage of patient tumors, and it should also be able to cause the same type of tumor to form in mice. No gene that fits either of these criteria has ever been found for ileal neuroendocrine tumors (I-NETs), which in humans are known for an unusual lack of recurrently mutated genes, and which have never been detected in mice. In the following report, we show that I-NETs can be generated by transgenic RT2 mice, which is a classic model for a genetically unrelated disease, pancreatic neuroendocrine tumors (PNETs). The ability of RT2 mice to generate I-NETs depended upon genetic background. I-NETs appeared in a B6AF1 genetic background, but not in a B6 background nor even in an AB6F1 background. AB6F1 and B6AF1 have identical nuclear DNA but can potentially express different allelic forms of imprinted genes. This led us to test human I-NETs for loss of imprinting, and we discovered that the IGF2 gene showed loss of imprinting and increased expression in the I-NETs of 57% of patients. By increasing IGF2 activity genetically, I-NETs could be produced by RT2 mice in a B6 genetic background, which otherwise never developed I-NETs. The facts that IGF2 is altered in a high percentage of patients with I-NETs and that I-NETs can form in mice that have elevated IGF2 activity, define IGF2 as the first genetic driver of ileal neuroendocrine tumorigenesis.

Alleles of Insm1 determine whether RIP1-Tag2 mice produce insulinomas or nonfunctioning pancreatic neuroendocrine tumors.

The two most common types of pancreatic neuroendocrine tumors (PanNETs) are insulinomas and nonfunctioning PanNETs (NF-PanNETs). Insulinomas are small, rarely metastatic tumors that secrete high amounts of insulin, and nonfunctioning PanNETs are larger tumors that are frequently metastatic but that do not secrete hormones. Insulinomas are modeled by the highly studied RIP1-Tag2 (RT2) transgenic mice when bred into a C57Bl/6 (B6) genetic background (also known as RT2 B6 mice). But there has been a need for an animal model of nonfunctioning PanNETs, which in the clinic are a more common and severe disease. Here we show that when bred into a hybrid AB6F1 genetic background, RT2 mice make nonfunctioning PanNETs. Compared to insulinomas produced by RT2 B6 mice, the tumors produced by RT2 AB6F1 mice were larger and more metastatic, and the animals did not suffer from hypoglycemia or hyperinsulinemia. Genetic crosses revealed that a locus in mouse chromosome 2qG1 was linked to liver metastasis and to lack of insulin production. This locus was tightly linked to the gene encoding Insm1, a beta cell transcription factor that was highly expressed in human insulinomas but unexpressed in other types of PanNETs due to promoter hypermethylation. Insm1-deficient human cell lines expressed stem cell markers, were more invasive in vitro, and metastasized at higher rates in vivo when compared to isogenic Insm1-expressing cell lines. These data demonstrate that expression of Insm1 can determine whether a PanNET is a localized insulinoma or a metastatic nonfunctioning tumor.

Sexual dimorphism of liver metastasis by murine pancreatic neuroendocrine tumors is affected by expression of complement C5.

In a mouse model for neuroendocrine tumors of the pancreas (PanNETs), liver metastasis occurred at a higher frequency in males. Male mice also had higher serum and intratumoral levels of the innate immunity protein complement C5. In mice that lost the ability to express complement C5, there was a lower frequency of metastasis, and males no longer had a higher frequency of metastasis than females. Treatment with PMX53, a small molecule antagonist of C5aR1/CD88, the receptor for complement C5a, also reduced metastasis. Mice lacking a functional gene for complement C5 had smaller primary tumors, which were less invasive and lacked the CD68+ macrophages that have previously been associated with metastasis in this type of tumor. This is the first report of a gene that causes sexual dimorphism of metastasis in a mouse model. In the human disease, which also shows sexual dimorphism for metastasis, clinically advanced tumors expressed more complement C5 than less advanced tumors.

Somatic mutation of CDKN1B in small intestine neuroendocrine tumors.

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and genome-sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NETs.

Attenuation of the retinoblastoma pathway in pancreatic neuroendocrine tumors due to increased cdk4/cdk6.

PURPOSE: In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs. EXPERIMENTAL DESIGN: Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines. RESULTS: A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway. CONCLUSIONS: Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials.

p53 negatively regulates transcription of the pyruvate dehydrogenase kinase Pdk2.

In cancer cells, the aberrant conversion of pyruvate into lactate instead of acetyl-CoA in the presence of oxygen is known as the Warburg effect. The consequences and mechanisms of this metabolic peculiarity are incompletely understood. Here we report that p53 status is a key determinant of the Warburg effect. Wild-type p53 expression decreased levels of pyruvate dehydrogenase kinase-2 (Pdk2) and the product of its activity, the inactive form of the pyruvate dehydrogenase complex (P-Pdc), both of which are key regulators of pyruvate metabolism. Decreased levels of Pdk2 and P-Pdc in turn promoted conversion of pyruvate into acetyl-CoA instead of lactate. Thus, wild-type p53 limited lactate production in cancer cells unless Pdk2 could be elevated. Together, our results established that wild-type p53 prevents manifestation of the Warburg effect by controlling Pdk2. These findings elucidate a new mechanism by which p53 suppresses tumorigenesis acting at the level of cancer cell metabolism.

Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans.

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d x 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

Osteoclasts resorb protein-free mineral (Osteologic discs) efficiently in the absence of osteopontin.

Osteopontin (OPN) is both a matrix protein in mineralized tissues and a cytokine, and it has a pivotal role in osteoclast-mediated bone resorption. Here, using a proprietary hydroxyapatite substitute for bone mineral (Osteologic discs), we investigated the requirement for OPN in mineral resorption. Resorption pits formed in the Osteologic discs, revealed by staining with silver nitrite (Von Kossa stain), were analyzed using the NIH Image J program, which can determine the number of pits formed per unit area, their average size, and the fractional area resorbed. After a preincubation of bone marrow cells from OPN -/- and OPN +/+ mice with M-CSF to allow the multiplication of osteoclast precursors on cell culture plastic, osteoclast formation on both Osteologic discs and standard cell culture plates was induced with soluble receptor activator of NFkappaB ligand, sRANKL. We did not detect a dramatic difference in osteoclast formation between OPN +/+ and OPN -/- cells, as judged by staining for tartrate-resistant acid phosphatase in osteoclasts formed on cell culture plastic, nor was there a significant difference in the ability of the osteoclasts to form resorption pits in the Osteologic discs. Additionally, none of six different anti-OPN monoclonal antibodies had a significant and reproducible effect on the formation or subsequent functioning of the OPN+/+ osteoclasts. These studies suggest that, in contrast to what has been found for normal bone, the efficiency of dissolution of a ceramic, protein-free (excepting protein adsorbed from the culture medium) hydroxyapatite/tri-calcium phosphate substrate by osteoclasts is not substantially enhanced by endogenous or exogenous OPN.