Search

Activating KRAS mutations (KRAS*) in pancreatic ductal adenocarcinoma (PDAC) drive anabolic metabolism and support tumor maintenance. KRAS* inhibitors show initial antitumor activity followed by recurrence due to cancer cell-intrinsic and immune-mediated paracrine mechanisms. Here, we explored the potential role of cancer-associated fibroblasts (CAFs) in enabling KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS*-independent growth is supported. Genetic extinction or pharmacological inhibition of KRAS* resulted in up-regulation of ERBB2 and ERBB3 expression in human and murine models, which prompted cancer cell utilization of CAF-derived NRG1 as a survival factor. Genetic depletion or pharmacological inhibition of ERBB2/3 or NRG1 abolished KRAS* bypass and synergized with KRAS G12D inhibitors in combination treatments in mouse and human PDAC models. Thus, we found that CAFs can contribute to KRAS* inhibitor therapy resistance via paracrine mechanisms, providing an actionable therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients., (© 2023 Han et al.; Published by Cold Spring Harbor Laboratory Press.)

Activating mutations in KRAS (KRAS*) are present in nearly all pancreatic ductal adenocarcinoma (PDAC) cases and critical for tumor maintenance. By using an inducible KRAS* PDAC mouse model, we identified a deubiquitinase USP21-driven resistance mechanism to anti-KRAS* therapy. USP21 promotes KRAS*-independent tumor growth via its regulation of MARK3-induced macropinocytosis, which serves to maintain intracellular amino acid levels for anabolic growth. The USP21-mediated KRAS* bypass, coupled with the frequent amplification of USP21 in human PDAC tumors, encourages the assessment of USP21 as a novel drug target as well as a potential parameter that may affect responsiveness to emergent anti-KRAS* therapy., (© 2021 Hou et al.; Published by Cold Spring Harbor Laboratory Press.)

Colorectal cancer has served as a genetic and biological paradigm for the evolution of solid tumors, and these insights have illuminated early detection, risk stratification, prevention, and treatment principles. Employing the hallmarks of cancer framework, we provide a conceptual framework to understand how genetic alterations in colorectal cancer drive cancer cell biology properties and shape the heterotypic interactions across cells in the tumor microenvironment. This review details research advances pertaining to the genetics and biology of colorectal cancer, emerging concepts gleaned from immune and single-cell profiling, and critical advances and remaining knowledge gaps influencing the development of effective therapies for this cancer that remains a major public health burden., (© 2021 Li et al.; Published by Cold Spring Harbor Laboratory Press.)

The ubiquitin-specific protease (USP) family is the largest group of cysteine proteases. Cancer genomic analysis identified frequent amplification of USP21 (22%) in human pancreatic ductal adenocarcinoma (PDAC). USP21 overexpression correlates with human PDAC progression, and enforced expression of USP21 accelerates murine PDAC tumor growth and drives PanIN to PDAC progression in immortalized human pancreatic ductal cells. Conversely, depletion of USP21 impairs PDAC tumor growth. Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness. Our work identifies and validates USP21 as a PDAC oncogene, providing a potential druggable target for this intractable disease., (© 2019 Hou et al.; Published by Cold Spring Harbor Laboratory Press.)

Despite the high long-term survival in localized prostate cancer, metastatic prostate cancer remains largely incurable even after intensive multimodal therapy. The lethality of advanced disease is driven by the lack of therapeutic regimens capable of generating durable responses in the setting of extreme tumor heterogeneity on the genetic and cell biological levels. Here, we review available prostate cancer model systems, the prostate cancer genome atlas, cellular and functional heterogeneity in the tumor microenvironment, tumor-intrinsic and tumor-extrinsic mechanisms underlying therapeutic resistance, and technological advances focused on disease detection and management. These advances, along with an improved understanding of the adaptive responses to conventional cancer therapies, anti-androgen therapy, and immunotherapy, are catalyzing development of more effective therapeutic strategies for advanced disease. In particular, knowledge of the heterotypic interactions between and coevolution of cancer and host cells in the tumor microenvironment has illuminated novel therapeutic combinations with a strong potential for more durable therapeutic responses and eventual cures for advanced disease. Improved disease management will also benefit from artificial intelligence-based expert decision support systems for proper standard of care, prognostic determinant biomarkers to minimize overtreatment of localized disease, and new standards of care accelerated by next-generation adaptive clinical trials., (© 2018 Wang et al.; Published by Cold Spring Harbor Laboratory Press.)

SMAD4 constrains progression of Pten -null prostate cancer and serves as a common downstream node of transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) pathways. Here, we dissected the roles of TGFβ receptor II (TGFBR2) and BMP receptor II (BMPR2) using a Pten -null prostate cancer model. These studies demonstrated that the molecular actions of TGFBR2 result in both SMAD4-dependent constraint of proliferation and SMAD4-independent activation of apoptosis. In contrast, BMPR2 deletion extended survival relative to Pten deletion alone, establishing its promoting role in BMP6-driven prostate cancer progression. These analyses reveal the complexity of TGFβ-BMP signaling and illuminate potential therapeutic targets for prostate cancer., (© 2018 Lu et al.; Published by Cold Spring Harbor Laboratory Press.)

Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the KRAS gene. To understand the role of oncogenic KRAS in CRC, we engineered a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele ( Kras mut ) and conditional null alleles of Apc and Trp53 (iKAP). The iKAP model recapitulates tumor progression from adenoma through metastases. Whole-exome sequencing revealed that the Kras mut allele was heterogenous in primary tumors yet homogenous in metastases, a pattern consistent with activated Kras mut signaling being a driver of progression to metastasis. System-level and functional analyses revealed the TGF-β pathway as a key mediator of Kras mut -driven invasiveness. Genetic extinction of Kras mut resulted in specific elimination of the Kras mut subpopulation in primary and metastatic tumors, leading to apoptotic elimination of advanced invasive and metastatic disease . This faithful CRC model provides genetic evidence that Kras mut drives CRC invasion and maintenance of metastases., (© 2017 Boutin et al.; Published by Cold Spring Harbor Laboratory Press.)

With 5-year survival rates remaining constant at 6% and rising incidences associated with an epidemic in obesity and metabolic syndrome, pancreatic ductal adenocarcinoma (PDAC) is on track to become the second most common cause of cancer-related deaths by 2030. The high mortality rate of PDAC stems primarily from the lack of early diagnosis and ineffective treatment for advanced tumors. During the past decade, the comprehensive atlas of genomic alterations, the prominence of specific pathways, the preclinical validation of such emerging targets, sophisticated preclinical model systems, and the molecular classification of PDAC into specific disease subtypes have all converged to illuminate drug discovery programs with clearer clinical path hypotheses. A deeper understanding of cancer cell biology, particularly altered cancer cell metabolism and impaired DNA repair processes, is providing novel therapeutic strategies that show strong preclinical activity. Elucidation of tumor biology principles, most notably a deeper understanding of the complexity of immune regulation in the tumor microenvironment, has provided an exciting framework to reawaken the immune system to attack PDAC cancer cells. While the long road of translation lies ahead, the path to meaningful clinical progress has never been clearer to improve PDAC patient survival., (© 2016 Ying et al.; Published by Cold Spring Harbor Laboratory Press.)

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity., (© 2014 Zheng et al.; Published by Cold Spring Harbor Laboratory Press.)

Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45β--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFβR2 and the TGFβ signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFβ pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.

Glioblastoma is both the most common and lethal primary malignant brain tumor. Extensive multiplatform genomic characterization has provided a higher-resolution picture of the molecular alterations underlying this disease. These studies provide the emerging view that "glioblastoma" represents several histologically similar yet molecularly heterogeneous diseases, which influences taxonomic classification systems, prognosis, and therapeutic decisions.

Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by intense apoptosis resistance and extensive necrosis. Bcl2L12 (for Bcl2-like 12) is a cytoplasmic and nuclear protein that is overexpressed in primary GBM and functions to inhibit post-mitochondrial apoptosis signaling. Here, we show that nuclear Bcl2L12 physically and functionally interacts with the p53 tumor suppressor, as evidenced by the capacity of Bcl2L12 to (1) enable bypass of replicative senescence without concomitant loss of p53 or p19 (Arf), (2) inhibit p53-dependent DNA damage-induced apoptosis, (3) impede the capacity of p53 to bind some of its target gene promoters, and (4) attenuate endogenous p53-directed transcriptomic changes following genotoxic stress. Correspondingly, The Cancer Genome Atlas profile and tissue protein analyses of human GBM specimens show significantly lower Bcl2L12 expression in the setting of genetic p53 pathway inactivation. Thus, Bcl2L12 is a multifunctional protein that contributes to intense therapeutic resistance of GBM through its ability to operate on two key nodes of cytoplasmic and nuclear signaling cascades.

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Cellular origins and genetic factors governing the genesis and maintenance of glioblastomas (GBM) are not well understood. Here, we report a pathogenetic role of the developmental regulator Id4 (inhibitor of differentiation 4) in GBM. In primary murine Ink4a/Arf(-/-) astrocytes, and human glioma cells, we provide evidence that enforced Id4 can drive malignant transformation by stimulating increased cyclin E to produce a hyperproliferative profile and by increased Jagged1 expression with Notch1 activation to drive astrocytes into a neural stem-like cell state. Thus, Id4 plays an integral role in the transformation of astrocytes via its combined actions on two-key cell cycle and differentiation regulatory molecules.

Activation of the transcription factor STAT3 is thought to potently promote oncogenesis in a variety of tissues, leading to intense efforts to develop STAT3 inhibitors for many tumors, including the highly malignant brain tumor glioblastoma. However, the function of STAT3 in glioblastoma pathogenesis has remained unknown. Here, we report that STAT3 plays a pro-oncogenic or tumor-suppressive role depending on the mutational profile of the tumor. Deficiency of the tumor suppressor PTEN triggers a cascade that inhibits STAT3 signaling in murine astrocytes and human glioblastoma tumors. Specifically, we forge a direct link between the PTEN-Akt-FOXO axis and the leukemia inhibitory factor receptor beta (LIFRbeta)-STAT3 signaling pathway. Accordingly, PTEN knockdown induces efficient malignant transformation of astrocytes upon knockout of the STAT3 gene. Remarkably, in contrast to the tumor-suppressive function of STAT3 in the PTEN pathway, STAT3 forms a complex with the oncoprotein epidermal growth factor receptor type III variant (EGFRvIII) in the nucleus and thereby mediates EGFRvIII-induced glial transformation. These findings indicate that STAT3 plays opposing roles in glial transformation depending on the genetic background of the tumor, providing the rationale for tailored therapeutic intervention in glioblastoma.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Malignant astrocytic gliomas such as glioblastoma are the most common and lethal intracranial tumors. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The recent confluence of advances in stem cell biology, cell signaling, genome and computational science and genetic model systems have revolutionized our understanding of the mechanisms underlying the genetics, biology and clinical behavior of glioblastoma. This progress is fueling new opportunities for understanding the fundamental basis for development of this devastating disease and also novel therapies that, for the first time, portend meaningful clinical responses.

Glioblastoma (GBM) is an astrocytic brain tumor characterized by an aggressive clinical course and intense resistance to all therapeutic modalities. Here, we report the identification and functional characterization of Bcl2L12 (Bcl2-like-12) that is robustly expressed in nearly all human primary GBMs examined. Enforced Bcl2L12 expression confers marked apoptosis resistance in primary cortical astrocytes, and, conversely, its RNA interference (RNAi)-mediated knockdown sensitizes human glioma cell lines toward apoptosis in vitro and impairs tumor growth with increased intratumoral apoptosis in vivo. Mechanistically, Bcl2L12 expression does not affect cytochrome c release or apoptosome-driven caspase-9 activation, but instead inhibits post-mitochondrial apoptosis signaling at the level of effector caspase activation. One of Bcl2L12's mechanisms of action stems from its ability to interact with and neutralize caspase-7. Notably, while enforced Bcl2L12 expression inhibits apoptosis, it also engenders a pronecrotic state, which mirrors the cellular phenotype elicited by genetic or pharmacologic inhibition of post-mitochondrial apoptosis molecules. Thus, Bcl2L12 contributes to the classical tumor biological features of GBM such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer.

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-beta (TGF-beta) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-beta-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-beta-dependent growth.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a median survival of <6 mo and a dismal 5-yr survival rate of 3%-5%. The cancer's lethal nature stems from its propensity to rapidly disseminate to the lymphatic system and distant organs. This aggressive biology and resistance to conventional and targeted therapeutic agents leads to a typical clinical presentation of incurable disease at the time of diagnosis. The well-defined serial histopathologic picture and accompanying molecular profiles of PDAC and its precursor lesions have provided the framework for emerging basic and translational research. Recent advances include insights into the cancer's cellular origins, high-resolution genomic profiles pointing to potential new therapeutic targets, and refined mouse models reflecting both the genetics and histopathologic evolution of human PDAC. This confluence of developments offers the opportunity for accelerated discovery and the future promise of improved treatment.

Werner Syndrome (WS) is characterized by premature aging, genomic instability, and cancer. The combined impact of WRN helicase deficiency and limiting telomere reserves is central to disease pathogenesis. Here, we report that cells doubly deficient for telomerase and WRN helicase show chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids. Somatic reconstitution of WRN function, but not a WRN helicase-deficient mutant, abolished telomere sister chromatid exchange (T-SCE), indicating that WRN normally represses T-SCEs. Elevated T-SCE was associated with greater immortalization potential and resultant tumors maintained telomeres via the alternative lengthening of telomere (ALT) pathway. We propose that the increased incidence of chromosomal instability and cancer in WS relates in part to aberrant recombinations between sister chromatids at telomeres, which facilitates the activation of ALT and engenders cancer-relevant chromosomal aberrations and tumor formation.

mSin3A is a core component of a large multiprotein corepressor complex with associated histone deacetylase (HDAC) enzymatic activity. Physical interactions of mSin3A with many sequence-specific transcription factors has linked the mSin3A corepressor complex to the regulation of diverse signaling pathways and associated biological processes. To dissect the complex nature of mSin3A's actions, we monitored the impact of conditional mSin3A deletion on the developmental, cell biological, and transcriptional levels. mSin3A was shown to play an essential role in early embryonic development and in the proliferation and survival of primary, immortalized, and transformed cells. Genetic and biochemical analyses established a role for mSin3A/HDAC in p53 deacetylation and activation, although genetic deletion of p53 was not sufficient to attenuate the mSin3A null cell lethal phenotype. Consistent with mSin3A's broad biological activities beyond regulation of the p53 pathway, time-course gene expression profiling following mSin3A deletion revealed deregulation of genes involved in cell cycle regulation, DNA replication, DNA repair, apoptosis, chromatin modifications, and mitochondrial metabolism. Computational analysis of the mSin3A transcriptome using a knowledge-based database revealed several nodal points through which mSin3A influences gene expression, including the Myc-Mad, E2F, and p53 transcriptional networks. Further validation of these nodes derived from in silico promoter analysis showing enrichment for Myc-Mad, E2F, and p53 cis-regulatory elements in regulatory regions of up-regulated genes following mSin3A depletion. Significantly, in silico promoter analyses also revealed specific cis-regulatory elements binding the transcriptional activator Stat and the ISWI ATP-dependent nucleosome remodeling factor Falz, thereby expanding further the mSin3A network of regulatory factors. Together, these integrated genetic, biochemical, and computational studies demonstrate the involvement of mSin3A in the regulation of diverse pathways governing many aspects of normal and neoplastic growth and survival and provide an experimental framework for the analysis of essential genes with diverse biological functions.

Lung cancer is the leading cause of cancer mortality worldwide. With the recent success of molecularly targeted therapies in this disease, a detailed knowledge of the spectrum of genetic lesions in lung cancer represents a critical step in the development of additional effective agents. An integrated high-resolution survey of regional amplifications and deletions and gene expression profiling of non-small-cell lung cancers (NSCLC) identified 93 focal high-confidence copy number alterations (CNAs), with 21 spanning less than 0.5 Mb with a median of five genes. Most CNAs were novel and included high-amplitude amplification and homozygous deletion events. Pathogenic relevance of these genomic alterations was further reinforced by their recurrence and overlap with focal alterations of other tumor types. Additionally, the comparison of the genomic profiles of the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC), showed an almost complete overlap with the exception of one amplified region on chromosome 3, specific for SCC. Among the few genes overexpressed within this amplicon was p63, a known regulator of squamous cell differentiation. These findings suggest that the AC and SCC subtypes may arise from a common cell of origin and they are driven to their distinct phenotypic end points by altered expression of a limited number of key genes such as p63.

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing.

Tuberous sclerosis complex (TSC) and Peutz-Jeghers syndrome (PJS) are dominantly inherited benign tumor syndromes that share striking histopathological similarities. Here we show that LKB1, the gene mutated in PJS, acts as a tumor suppressor by activating TSC2, the gene mutated in TSC. Like TSC2, LKB1 inhibits the phosphorylation of the key translational regulators S6K and 4EBP1. Furthermore, we show that LKB1 activates TSC2 through the AMP-dependent protein kinase (AMPK), indicating that LKB1 plays a role in cell growth regulation in response to cellular energy levels. Our results suggest that PJS and other benign tumor syndromes could be caused by dysregulation of the TSC2/mTOR pathway.

Pancreatic ductal adenocarcinoma ranks among the most lethal of human malignancies. Here, we assess the cooperative interactions of two signature mutations in mice engineered to sustain pancreas-specific Cre-mediated activation of a mutant Kras allele (KrasG12D) and deletion of a conditional Ink4a/Arf tumor suppressor allele. The phenotypic impact of KrasG12D alone was limited primarily to the development of focal premalignant ductal lesions, termed pancreatic intraepithelial neoplasias (PanINs), whereas the sole inactivation of Ink4a/Arf failed to produce any neoplastic lesions in the pancreas. In combination, KrasG12D expression and Ink4a/Arf deficiency resulted in an earlier appearance of PanIN lesions and these neoplasms progressed rapidly to highly invasive and metastatic cancers, resulting in death in all cases by 11 weeks. The evolution of these tumors bears striking resemblance to the human disease, possessing a proliferative stromal component and ductal lesions with a propensity to advance to a poorly differentiated state. These findings in the mouse provide experimental support for the widely accepted model of human pancreatic adenocarcinoma in which activated KRAS serves to initiate PanIN lesions, and the INK4A/ARF tumor suppressors function to constrain the malignant conversion of these PanIN lesions into lethal ductal adenocarcinoma. This faithful mouse model may permit the systematic analysis of genetic lesions implicated in the human disease and serve as a platform for the identification of early disease markers and for the efficient testing of novel therapies.

The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division. In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation. However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation. Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis. mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes. Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes. On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells.

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(-/-) Ink4a/Arf(-/-) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(-/-) Ink4a/Arf(-/-) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(-/-) Ink4a/Arf(-/-) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the basis for the evolutionary conservation of myc gene family has remained unclear. To elucidate this issue, we have generated mice in which the endogenous c-myc coding sequences have been replaced with N-myc coding sequences. Strikingly, mice homozygous for this replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have evolved, to a large extent, to facilitate differential patterns of expression.

Loss of Max function in the mouse resulted in generalized developmental arrest of both embryonic and extraembryonic tissues at early postimplantation (approximately E5.5-6.5), coincident with loss or dilution of maternal Max stores in the expanding embryo in vivo and in blastocyst outgrowths in vitro. Developmentally arrested embryos were reduced in size and exhibited widespread cytological degeneration and feeble BrdU incorporation. Max and, by extension, the Myc superfamily, serve essential roles in early mammalian development and a maternal reservoir of Max exists in sufficient amount to sustain Myc superfamily function through preimplantation stages of development.

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%-50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a-ARF locus, which encodes two gene products (p16(INK4a) and p19(ARF)) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a-ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.

Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that the Cdk inhibitors p27(KIP1) and p57(KIP2) function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27(KIP1) and p57(KIP2) are critical terminal effectors of signal transduction pathways that control cell differentiation.

The familial melanoma gene (INK4a/MTS1/CDKN2) encodes potent tumor suppressor activity. Although mice null for the ink4a homolog develop a cancer-prone condition, a pathogenetic link to melanoma susceptibility has yet to be established. Here we report that mice with melanocyte-specific expression of activated H-rasG12V on an ink4a-deficient background develop spontaneous cutaneous melanomas after a short latency and with high penetrance. Consistent loss of the wild-type ink4a allele was observed in tumors arising in ink4a heterozygous transgenic mice. No homozygous deletion of the neighboring ink4b gene was detected. Moreover, as in human melanomas, the p53 gene remained in a wild-type configuration with no observed mutation or allelic loss. These results show that loss of ink4a and activation of Ras can cooperate to accelerate the development of melanoma and provide the first in vivo experimental evidence for a causal relationship between ink4a deficiency and the pathogenesis of melanoma. In addition, this mouse model affords a system in which to identify and analyze pathways involved in tumor progression against the backdrop of genetic alterations encountered in human melanomas.

The myc family of cellular oncogenes encodes three highly related nuclear phosphoproteins (c-Myc, N-Myc, and L-Myc) that are believed to function as sequence-specific transcription factors capable of regulating genes important in cellular growth and differentiation. Current evidence indicates that Myc family proteins exist as biologically active heterodimeric complexes in association with another helix-loop-helix leucine zipper phosphoprotein, Max. We have investigated the common and unique properties among the Myc family, as well as the physiological role of Max in the regulation of Myc family function. We demonstrate that trans-activation-incompetent mutants of one Myc family member can act in trans to dominantly suppress the cotransformation activities of all three Myc oncoproteins, indicating that the Myc family functions through common genetic elements in its cellular transformation pathways. Employing co-immunoprecipitation with either anti-Myc or anti-Max antibodies, we show that the transfected normal c-Myc, N-Myc, and L-Myc oncoproteins associate with the endogenous Max protein in REF transformants, indicating that the Max interaction represents at least one component common to Myc family function. In addition, we observed a striking reduction in Myc cotransformation activity when a Max expression construct was added to myc/ras co-transfections. We discuss these biological findings in the context of a proposed model for Myc/Max function and regulation in which Max serves as either an obligate partner in the Myc/Max transcriptional complex or as a repressor in the form of a transcriptionally inert Max/Max homodimer capable of occupying Myc/Max-responsive gene targets.

We have determined the nucleotide sequence and transforming activity of the human L-myc gene and a processed L-myc pseudogene (L-myc psi). We demonstrate by cotransformation assays that a 10.6-kb EcoRI fragment derived from a human placental library contains a complete and functional L-myc gene including transcriptional regulatory sequences sufficient for expression in rat embryo fibroblasts. Organization of the L-myc gene was determined by comparing its sequence to those of the L-myc psi gene and an L-myc cDNA clone derived from a human small cell lung carcinoma. Our results show that L-myc has a three-exon organization similar to that of the c-myc and N-myc genes. The putative L-myc gene product consists of 364 amino acids and contains five of the seven homology regions highly conserved between c-myc and N-myc. These conserved regions are located along the entire length of the putative L-myc protein and are interspersed among nonconserved regions. While the putative L-myc gene product is of a smaller size when compared to the c- and N-myc proteins, the relative positions of certain conserved residues occur in corresponding locations along the peptide backbone of the three proteins. In addition, comparison of the human and murine L-myc gene sequences indicate that the relatively large 5' and 3' untranslated regions are evolutionarily conserved, but that these sequences are totally divergent between the L-, c-, and N-myc genes. Finally, we demonstrate that, like the N- and c-myc genes, the L-myc gene can cooperate with a mutant Ha-ras gene to cause malignant transformation of rat embryo fibroblasts in culture. Our analyses clearly prove that L-myc represents a functional member of the myc oncogene family and further delineate structural features that may be important for the common and divergent functions of the members of this gene family.