[Show abstract][Hide abstract] ABSTRACT: Lung cancer in never-smokers is an important disease often characterized by mutations in epidermal growth factor receptor (EGFR), yet risk reduction measures and effective chemopreventive strategies have not been established. We identify mammalian target of rapamycin (mTOR) as potentially valuable target for EGFR mutant lung cancer. mTOR is activated in human lung cancers with EGFR mutations, and this increases with acquisition of T790M mutation. In a mouse model of EGFR mutant lung cancer, mTOR activation is an early event. As a single agent, the mTOR inhibitor rapamycin prevents tumor development, prolongs overall survival, and improves outcomes after treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI). These studies support clinical testing of mTOR inhibitors in order to prevent the development and progression of EGFR mutant lung cancers.
[Show abstract][Hide abstract] ABSTRACT: Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review.
[Show abstract][Hide abstract] ABSTRACT: Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.
[Show abstract][Hide abstract] ABSTRACT: Objectives
Primary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors.
Materials and Methods
Immunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed.
Results and Conclusion
Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFRL858R/T790M mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3 K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3 K/AKT inhibitors and expression of SOX2 was examined. PI3 K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3 K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.
Lung cancer (Amsterdam, Netherlands) 01/2014; · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metformin is the most commonly prescribed drug for type II diabetes and is associated with decreased cancer risk. Previously, we showed that metformin prevented tobacco carcinogen (NNK)-induced lung tumorigenesis in a non-diabetic mouse model, which was associated with decreased IGF-I/insulin receptor signaling but not activation of AMPK in lung tissues, as well as decreased circulating levels of IGF-1 and insulin. Here, we used liver-IGF-1-deficient (LID) mice to determine the importance of IGF-1 in NNK-induced lung tumorigenesis and chemoprevention by metformin. LID mice had decreased lung tumor multiplicity and burden compared to WT mice. Metformin further decreased lung tumorigenesis in LID mice without affecting IGF-1 levels, suggesting that metformin can act through IGF-1-independent mechanisms. In lung tissues, metformin decreased phosphorylation of multiple receptor tyrosine kinases (RTKs) as well as levels of GTP-bound Ras independently of AMPK. Metformin also diminished plasma levels of several cognate ligands for these RTKs. Tissue distribution studies using [14C]-metformin showed that uptake of metformin was high in liver but 4 fold lower in lungs, suggesting that the suppression of RTK activation by metformin occurs predominantly via systemic, indirect effects. Systemic inhibition of circulating growth factors and local RTK signaling are new AMPK-independent mechanisms of action of metformin that could underlie its ability to prevent tobacco carcinogen-induced lung tumorigenesis.
Cancer Prevention Research 06/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metformin is the most commonly prescribed drug for type 2 diabetes (T2DM). Retrospective studies show that metformin is associated with decreased cancer risk. This historical correlation has driven vigorous research campaigns to determine the anticancer mechanisms of metformin. Consolidating the preclinical data is a challenge because unanswered questions remain concerning relevant mechanisms, bioavailability, and genetic factors that confer metformin sensitivity. Perhaps the most important unanswered question is whether metformin has activity against cancer in non-diabetics. In this review we highlight the proposed mechanisms of metformin action in cancer and discuss ongoing clinical trials with metformin in cancer. Improved understanding of these issues will increase the chances for successful application of metformin as an inexpensive, well-tolerated, and effective anticancer agent.
Trends in Endocrinology and Metabolism 06/2013; · 8.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Cowden syndrome (CS) is a hereditary cancer syndrome associated with a germline mutation in PTEN.Patients are predisposed to multiple malignancies including renal cell carcinoma (RCC). METHODS: Patients with CS were evaluated as part of a clinical protocol. Those with a history of RCC underwent review of clinical features, tumor characteristics, and family history. Renal tumors were evaluated for loss of heterozygosity (LOH). RESULTS: Among 24 CS patients, 4 were identified with RCC (16.7%). Three patients had solitary tumors, two with papillary type I histology and one with clear cell histology. The fourth patient had bilateral, synchronous chromophobe tumors. No patients had a prior family history of RCC. All RCC patients had dermatologic manifestations of CS and had macrocephaly. LOH at the PTEN mutation was identified in 4 tumors (80%). No genotype-phenotype association was found, as the same mutation was identified in different RCC histologies. CONCLUSION: RCC is an underappreciated feature of CS. As most patients lack a prior family history or a distinctive RCC histology, recognition of the associated non-renal features should target referral for genetic counseling. PTEN LOH is common in CS renal tumors. Because loss of PTEN can activate mTOR and mTOR inhibitors are FDA-approved to treat RCC, these agents have clinical potential in RCC associated with CS.
The Journal of urology 06/2013; · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p38α mitogen-activated protein kinase (MAPK) is generally activated by dual phosphorylation but has also been shown to exhibit alternative activation modes. One of these modes included a direct interaction with phosphatidylinositol ether lipid analogues (PIA) inducing p38α autoactivation and apoptosis. Perifosine, an Akt inhibitor in phase II clinical trials, also showed p38α activation properties similarly to those of PIAs. The crystal structures of p38α in complex with PIA23, PIA24 and perifosine provide insights into this unique activation mode. The activating molecules bind a unique hydrophobic binding site in the kinase C'-lobe formed in part by the MAPK insert region. In addition, there are conformational changes in the short αEF/αF loop region that acts as an activation switch, inducing autophosphorylation. Structural and biochemical characterization of the αEF/αF loop identified Trp197 as a key residue in the lipid binding and in p38α catalytic activity. The lipid binding site also accommodates hydrophobic inhibitor molecules and, thus, can serve as a novel p38α-target for specific activation or inhibition, with novel therapeutic implications.
Journal of Molecular Biology 10/2012; · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Li-Fraumeni syndrome (LFS) is a rare dominantly inherited cancer predisposition syndrome that was first described in 1969. In most families, it is caused by germline mutations in the TP53 gene and is characterized by early onset of multiple specific cancers and very high lifetime cumulative cancer risk. Despite significant progress in understanding the molecular biology of TP53, the optimal clinical management of this syndrome is poorly defined. We convened a workshop on November 2, 2010, at the National Institutes of Health in Bethesda, Maryland, bringing together clinicians and scientists, as well as individuals from families with LFS, to review the state of the science, address clinical management issues, stimulate collaborative research, and engage the LFS family community. This workshop also led to the creation of the Li-Fraumeni Exploration (LiFE) Research Consortium.
Cancer Genetics 08/2012; 205(10):479-87. · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exploiting protein homeostasis is a new therapeutic approach in cancer. Nelfinavir (NFV) is an HIV protease inhibitor that induces endoplasmic reticulum (ER) stress in cancer cells. Under conditions of ER stress, misfolded proteins are transported from the ER back to the cytosol for subsequent degradation by the ubiquitin-proteasome system. Bortezomib (BZ) is a proteasome inhibitor and interferes with degradation of misfolded proteins. Here, we show that NFV and BZ enhance proteotoxicity in non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells. The combination synergistically inhibited cell proliferation and induced cell death. Activating transcription factor (ATF)3 and CCAAT-enhancer binding protein homologous protein (CHOP), markers of ER stress, were rapidly increased, and their siRNA-mediated knockdown inhibited cell death. Knockdown of double-stranded RNA activated protein kinase-like ER kinase, a signal transducer in ER stress, significantly decreased apoptosis. Pretreatment with the protein synthesis inhibitor, cycloheximide, decreased levels of ubiquitinated proteins, ATF3, CHOP, and the overall total cell death, suggesting that inhibition of protein synthesis increases cell survival by relieving proteotoxic stress. The NFV/BZ combination inhibited the growth of NSCLC xenografts, which correlated with the induction of markers of ER stress and apoptosis. Collectively, these data show that NFV and BZ enhance proteotoxicity in NSCLC and MM cells, and suggest that this combination could tip the precarious balance of protein homeostasis in cancer cells for therapeutic gain.
Cell Death & Disease 01/2012; 3:e353. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.
Cell Death & Disease 01/2012; 3:e340. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The remarkably heterogeneous nature of lung cancer has become more apparent over the last decade. In general, advanced lung cancer is an aggressive malignancy with a poor prognosis. The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer, however, has created new opportunities for targeted therapy and improved outcome. In this paper, we define "molecular subtypes" of lung cancer based on specific actionable genetic aberrations. Each subtype is associated with molecular tests that define the subtype and drugs that may potentially treat it. We hope this paper will be a useful guide to clinicians and researchers alike by assisting in therapy decision making and acting as a platform for further study. In this new era of cancer treatment, the 'one-size-fits-all' paradigm is being forcibly pushed aside-allowing for more effective, personalized oncologic care to emerge.
PLoS ONE 01/2012; 7(2):e31906. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.
Cancer Prevention Research 11/2011; 4(11):1743-51. · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lung tumors from smokers as well as lung tumors from mice exposed to tobacco carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), often carry mutations in K-ras, which activates downstream-signaling pathways such as PI3K/AKT/mTOR pathway. Mice with genetic deletion of one of three isoforms of AKT were used to investigate the role of AKT in mutant K-ras-induced lung tumorigenesis in mice. Although deletion of Akt1 or Akt2 decreased NNK-induced lung tumor formation by 90%, deletion of Akt2 failed to decrease lung tumorigenesis in two other mouse models driven by mutant K-ras. Genetic mapping showed that Akt2 was tightly linked to the cytochrome P450 Cyp2a locus on chromosome 7. Consequently, targeted deletion of Akt2 created linkage to a strain-specific Cyp2a5 polymorphism that decreased activation of NNK in vitro. Mice with this Cyp2a5 polymorphism had decreased NNK-induced DNA adduct formation in vivo and decreased NNK-induced lung tumorigenesis. These studies support human epidemiological studies linking CYP2A polymorphisms with lung cancer risk in humans and highlight the need to confirm phenotypes of genetically engineered mice in multiple mouse strains.
[Show abstract][Hide abstract] ABSTRACT: Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIA) are analogues of the products of phosphoinositide-3-kinase (PI3K) that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of five active PIAs and the PI3K inhibitor LY294002 (LY) was conducted in non-small cell lung carcinoma cells using high-density oligonucleotide arrays. Gene ontology analysis revealed that genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, whereas genes involved in DNA replication, repair, and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into three groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, and PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB, and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3, and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB), and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity.
Molecular Cancer Therapeutics 05/2011; 10(7):1137-48. · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PTEN is among the most frequently inactivated tumour suppressor genes in sporadic cancer. PTEN has dual protein and lipid phosphatase activity, and its tumour suppressor activity is dependent on its lipid phosphatase activity, which negatively regulates the PI3K-AKT-mTOR pathway. Germline mutations in PTEN have been described in a variety of rare syndromes that are collectively known as the PTEN hamartoma tumour syndromes (PHTS). Cowden syndrome is the best-described syndrome within PHTS, with approximately 80% of patients having germline PTEN mutations. Patients with Cowden syndrome have an increased incidence of cancers of the breast, thyroid and endometrium, which correspond to sporadic tumour types that commonly exhibit somatic PTEN inactivation. Pten deletion in mice leads to Cowden syndrome-like phenotypes, and tissue-specific Pten deletion has provided clues to the role of PTEN mutation and loss in specific tumour types. Studying PTEN in the continuum of rare syndromes, common cancers and mouse models provides insight into the role of PTEN in tumorigenesis and will inform targeted drug development.
Nature Reviews Cancer 04/2011; 11(4):289-301. · 29.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery.
PLoS ONE 01/2011; 6(8):e23849. · 3.53 Impact Factor