SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC-alone or in combination with PDAC-relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRAS G12D allele, SMAD4 deficiency enabled rapid progression of KRAS G12D -initiated neoplasms. While KRAS G12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRAS G12D and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRAS G12D INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-␤ (TGF-␤) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-␤-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRAS G12D -initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-␤-dependent growth.[Keywords: Smad4; pancreatic cancer; epithelial-to-mesenchymal transition mouse models; TGF-␤] Supplemental material is available at http://www.genesdev.org. PDAC (pancreatic ductal adenocarcinoma) ranks as the fourth leading cause of cancer mortality in the United States and carries a median survival of <6 mo (Li et al. 2004). Hallmarks of this disease include the infiltration of the tumor with a proliferative stroma (desmoplasia), early invasion and metastasis, and pronounced genomic instability (Solcia et al. 1995). PDAC is characterized by a highly recurrent pattern of genetic lesions consisting of activating mutations of KRAS and inactivation of INK4A (via mutation, deletion, or promoter methylation) in virtually all cases, inactivation of the p53-ARF pathway in ∼87% of cases (including tumors with deletions of the INK4A/ARF locus), and SMAD4 inactivation in ∼53% (Hansel et al. 2003). Hence, SMAD4 status can be considered as a distinguishing molecular feature of two major classes of PDAC. Significant ongoing efforts are being directed toward the elucidation of how specific signature mutations contribute to the genesis and progression of PDAC and influence its tumor biological features.

BackgroundPancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.Methods and FindingsHere, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.ConclusionsOur approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.

Lkb1 is a central regulator of cell polarity and energy metabolism through its capacity to activate the AMP-activated protein kinase (AMPK)-related family of protein kinases. Germ line-inactivating mutation of Lkb1 leads to Peutz-Jeghers syndrome, which is characterized by benign hamartomas and a susceptibility to malignant epithelial tumors. Mutations in Lkb1 are also found in sporadic carcinomas, most frequently in lung cancers associated with tobacco carcinogen exposure. The basis for Lkb1-dependent tumor suppression is not defined. Here, we uncover a marked sensitivity of Lkb1 mutant mice to the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Lkb1 +/À mice are highly prone to DMBAinduced squamous cell carcinoma (SCC) of the skin and lung. Confirming a cell autonomous tumor suppressor role of Lkb1, mice with epidermal-specific Lkb1 deletion are also susceptible to DMBA-induced SCC and develop spontaneous SCC with long latency. Restoration of wild-type Lkb1 causes senescence in tumor-derived cell lines, a process that can be partially bypassed by inactivation of the Rb pathway, but not by inactivation of p53 or AMPK. Our data indicate that Lkb1 is a potent suppressor of carcinogen-induced skin and lung cancers and that downstream targets beyond the AMPK-mTOR pathway are likely mediators of Lkb1-dependent tumor suppression.

The metabolic adaptations to fasting in the liver are largely controlled by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARα), where PPARα upregulates genes encoding the biochemical pathway for β-oxidation of fatty acids and ketogenesis. As part of an effort to identify and characterize nutritionally regulated genes that play physiological roles in the adaptation to fasting, we identified Major facilitator superfamily domain-containing protein 2a (Mfsd2a) as a fasting-induced gene regulated by both PPARα and glucagon signaling in the liver. MFSD2A is a cell-surface protein homologous to bacterial sodium-melibiose transporters. Hepatic expression and turnover of MFSD2A is acutely regulated by fasting/refeeding, but expression in the brain is constitutive. Relative to wildtype mice, gene-targeted Mfsd2a knockout mice are smaller, leaner, and have decreased serum, liver and brown adipose triglycerides. Mfsd2a knockout mice have normal liver lipid metabolism but increased whole body energy expenditure, likely due to increased β-oxidation in brown adipose tissue and significantly increased voluntary movement, but surprisingly exhibited a form of ataxia. Together, these results indicate that MFSD2A is a nutritionally regulated gene that plays myriad roles in body growth and development, motor function, and lipid metabolism. Moreover, these data suggest that the ligand(s) that are transported by MFSD2A play important roles in these physiological processes and await future identification.

Neonatal heart failure is a rare, poorly-understood presentation of familial dilated cardiomyopathy (DCM). Exome sequencing in a neonate with severe DCM revealed a homozygous nonsense variant in leiomodin 2 (LMOD2, p.Trp398*). Leiomodins (Lmods) are actin-binding proteins that regulate actin filament assembly. While disease-causing mutations in smooth (LMOD1) and skeletal (LMOD3) muscle isoforms have been described, the cardiac (LMOD2) isoform has not been previously associated with human disease. Like our patient, Lmod2-null mice have severe early-onset DCM and die before weaning. The infant’s explanted heart showed extraordinarily short thin filaments with isolated cardiomyocytes displaying a large reduction in maximum calcium-activated force production. The lack of extracardiac symptoms in Lmod2-null mice, and remarkable morphological and functional similarities between the patient and mouse model informed the decision to pursue cardiac transplantation in the patient. To our knowledge, this is the first report of aberrant cardiac thin filament assembly associated with human cardiomyopathy.

The INK4/ARF locus encodes the p15(INK4B), p16(INK4A) and p14(ARF) tumor suppressor proteins whose loss of function is associated with the pathogenesis of many human cancers. Dissecting the relative contribution of these genes to growth control in vivo is complicated by their physical contiguity and the frequency of homozygous deletions that inactivate all three components of this locus. While genetically engineered mouse models provide a rigorous system for elucidating cancer gene function, there is some evidence to suggest there are cross-species differences in regulating tumor biology. Given the prevalence of mouse models in cancer research and the potential contribution of such models to preclinical studies, it is important determine to what degree the function of these critical tumor suppressors is conserved between organisms. In this review, we assess the relative biological roles of INK4A, INK4B and ARF in mice and humans with the aim of determining the faithfulness of mouse models and also of obtaining insights into the pattern of specific tumor types that are associated with germline and somatic mutations at components of this locus. We will discuss 1) the contribution of INK4A, INK4B and ARF to growth control in vitro in a series of cell types, 2) the in vivo phenotypes associated with germline loss of function of this locus and 3) the study of Ink4a and Arf in different cancer-specific mouse models.

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Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the Muscular Lamin A/C-interacting protein MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients’ skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans, and solidifies MLIP's role in normal and diseased skeletal muscle homeostasis.