ABSTRACT
T cell acute lymphoblastic leukemia (TALL), a neoplasm derived from T cell lineagecommitted lymphoblasts, is characterized by genetic alterations that result in activation of oncogenic transcription factors and the NOTCH1 pathway activation. The NOTCH is a transmembrane receptor protein activated by γsecretase. γsecretase inhibitors (GSIs) are a NOTCHtargeted therapy for TALL. However, their clinical application has not been successful due to adverse events (primarily gastrointestinal toxicity), limited efficacy, and drug resistance caused by several mechanisms, including activation of the AKT/mTOR pathway. Nelfinavir is an human immunodeficiency virus 1 aspartic protease inhibitor and has been repurposed as an anticancer drug. It acts by inducing endoplasmic reticulum (ER) stress and inhibiting the AKT/mTOR pathway. Thus, it was hypothesized that nelfinavir might inhibit the NOTCH pathway via γsecretase inhibition and blockade of aspartic protease presenilin, which would make nelfinavir effective against NOTCHassociated TALL. The present study assessed the efficacy of nelfinavir against TALL cells and investigated mechanisms of action in vitro and in preclinical treatment studies using a SCLLMO1 transgenic mouse model. Nelfinavir blocks presenilin 1 processing and inhibits γsecretase activity as well as the NOTCH1 pathway, thus suppressing TALL cell viability. Additionally, microarray analysis of nelfinavirtreated TALL cells showed that nelfinavir upregulated mRNA levels of CHAC1 (glutathionespecific γglutamylcyclotransferase 1, a negative regulator of NOTCH) and sestrin 2 (SESN2; a negative regulator of mTOR). As both factors are upregulated by ER stress, this confirmed that nelfinavir induced ER stress in TALL cells. Moreover, nelfinavir suppressed NOTCH1 mRNA expression in microarray analyses. These findings suggest that nelfinavir inhibited the NOTCH1 pathway by downregulating NOTCH1 mRNA expression, upregulating CHAC1 and suppressing γsecretase via presenilin 1 inhibition and the mTOR pathway by upregulating SESN2 via ER stress induction. Further, nelfinavir exhibited therapeutic efficacy against TALL in an SCLLMO1 transgenic mouse model. Collectively, these findings highlight the potential of nelfinavir as a novel therapeutic candidate for treatment of patients with TALL.
Subject(s)
Nelfinavir , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Mice , Animals , Nelfinavir/pharmacology , Nelfinavir/therapeutic use , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Presenilin-1 , Proto-Oncogene Proteins c-akt/metabolism , Amyloid Precursor Protein Secretases , TOR Serine-Threonine Kinases/metabolism , Enzyme Inhibitors , Transcription Factors , Mice, Transgenic , RNA, Messenger , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Cell Line, Tumor , SestrinsABSTRACT
BACKGROUND: The influence of anthropometric characteristics on colorectal neoplasia biology is unclear. We conducted a systematic review and meta-analysis to determine if adult-attained height is independently associated with the risk of colorectal cancer or adenoma. METHODS: We searched MEDLINE, EMBASE, the Cochrane Library, and Web of Science from inception to August 2020 for studies on the association between adult-attained height and colorectal cancer or adenoma. The original data from the Johns Hopkins (Baltimore, MD) Colon Biofilm study was also included. The overall HR/OR of colorectal cancer/adenoma with increased height was estimated using random-effects meta-analysis. RESULTS: We included 47 observational studies involving 280,644 colorectal cancer and 14,139 colorectal adenoma cases. Thirty-three studies reported data for colorectal cancer incidence per 10-cm increase in height; 19 yielded an HR of 1.14 [95% confidence interval (CI), 1.11-1.17; P < 0.001), and 14 engendered an OR of 1.09 (95% CI, 1.05-1.13; P < 0.001). Twenty-six studies compared colorectal cancer incidence between individuals within the highest versus the lowest height percentile; 19 indicated an HR of 1.24 (95% CI, 1.19-1.30; P < 0.001), and seven resulting in an OR of 1.07 (95% CI, 0.92-1.25; P = 0.39). Four studies reported data for assessing colorectal adenoma incidence per 10-cm increase in height, showing an overall OR of 1.06 (95% CI, 1.00-1.12; P = 0.03). CONCLUSIONS: Greater adult attained height is associated with an increased risk of colorectal cancer and adenoma. IMPACT: Height should be considered as a risk factor for colorectal cancer screening.
Subject(s)
Adenoma , Colorectal Neoplasms , Adenoma/prevention & control , Adult , Cohort Studies , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/etiology , Colorectal Neoplasms/prevention & control , Humans , Incidence , Risk FactorsABSTRACT
Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Animals , Fecal Microbiota Transplantation , Humans , Mice , Neoadjuvant Therapy , RNA, Ribosomal, 16S/genetics , Reproducibility of ResultsABSTRACT
Studies have found a positive association between metabolic risk factors, such as obesity and diabetes, and adenomatous polyps (AP). However, fewer studies have assessed the association between sessile serrated polyps (SSP) or synchronous diagnosis of APs and SSPs (synch polyps). Study participants (N = 1,370; ages 40-85) undergoing screening colonoscopy were enrolled between August 2016 and February 2020. Self-reported metabolic risk factors, including diabetes, hypertension, hyperlipidemia, and overweight/obesity, were evaluated for associations with new diagnoses of APs, SSPs, and synch polyps at the present colonoscopy. Average participant age was 60.73 ± 8.63 (SD) years; 56.7% were female and 90.9% white. In an assessment of individual metabolic risk factors, adjusted for age, sex, race, and smoking status, increased body mass index (BMI; overweight or obese vs. normal BMI of <25 kg/m2) was associated with an increased odds for new onset of colon APs (P trend < 0.001) as was a diagnosis of diabetes [adjusted conditional OR (aCOR) = 1.59 (1.10-2.29)]. No associations were seen between the metabolic risk factors and onset of SSPs. Being obese or hypertensive each increased the odds of new onset of synch polyps with aCOR values of 2.09 (1.01-4.32) and 1.79 (1.06-3.02), respectively. Self-reported risk factors may help assess polyp type risk. Because SSPs and synch polyps are rare, larger studies are needed to improve our understanding of the contribution of these factors to polyp risk. These data lead us to hypothesize that differences in observed metabolic risk factors between polyp types reflect select metabolic impact on pathways to colorectal cancer. PREVENTION RELEVANCE: Self-reported medical history provides valuable insight into polyp risk, potentially enabling the use of larger retrospective studies of colonoscopy populations to assess knowledge gaps. More aggressive colonoscopy screening, critical to colorectal cancer prevention, may be considered in populations of individuals with metabolic risk factors and modifiable lifestyle risk factors.
Subject(s)
Adenomatous Polyps/epidemiology , Colonic Polyps/epidemiology , Colorectal Neoplasms/prevention & control , Adenomatous Polyps/diagnosis , Adenomatous Polyps/metabolism , Adenomatous Polyps/pathology , Adult , Aged , Aged, 80 and over , Colonic Polyps/diagnosis , Colonic Polyps/metabolism , Colonic Polyps/pathology , Colonoscopy , Colorectal Neoplasms/pathology , Diabetes Mellitus/epidemiology , Diabetes Mellitus/metabolism , Female , Humans , Hyperlipidemias/epidemiology , Hyperlipidemias/metabolism , Hypertension/epidemiology , Hypertension/metabolism , Male , Medical History Taking/statistics & numerical data , Middle Aged , Obesity/epidemiology , Obesity/metabolism , Prospective Studies , Risk Factors , Self Report/statistics & numerical dataABSTRACT
PURPOSE: While immune checkpoint inhibitors (ICI) have revolutionized the treatment of cancer by producing durable antitumor responses, only 10%-30% of treated patients respond and the ability to predict clinical benefit remains elusive. Several studies, small in size and using variable analytic methods, suggest the gut microbiome may be a novel, modifiable biomarker for tumor response rates, but the specific bacteria or bacterial communities putatively impacting ICI responses have been inconsistent across the studied populations. EXPERIMENTAL DESIGN: We have reanalyzed the available raw 16S rRNA amplicon and metagenomic sequencing data across five recently published ICI studies (n = 303 unique patients) using a uniform computational approach. RESULTS: Herein, we identify novel bacterial signals associated with clinical responders (R) or nonresponders (NR) and develop an integrated microbiome prediction index. Unexpectedly, the NR-associated integrated index shows the strongest and most consistent signal using a random effects model and in a sensitivity and specificity analysis (P < 0.01). We subsequently tested the integrated index using validation cohorts across three distinct and diverse cancers (n = 105). CONCLUSIONS: Our analysis highlights the development of biomarkers for nonresponse, rather than response, in predicting ICI outcomes and suggests a new approach to identify patients who would benefit from microbiome-based interventions to improve response rates.
Subject(s)
Biomarkers , Computational Biology , Immune Checkpoint Inhibitors/pharmacology , Microbiota/drug effects , Bacteria/classification , Bacteria/genetics , Computational Biology/methods , Gastrointestinal Microbiome , Genome, Bacterial , Humans , Immune Checkpoint Inhibitors/therapeutic use , Metagenomics/methods , Microbiota/genetics , RNA, Ribosomal, 16S , ROC Curve , Reproducibility of Results , Whole Genome SequencingABSTRACT
BACKGROUND/AIM: Nelfinavir is a human immunodeficiency virus protease inhibitor that is currently being repositioned as an anticancer drug. Chloroquine, an anti-malarial lysosomotropic drug, inhibits autophagy. It has been reported that the combination of nelfinavir and chloroquine significantly enhances endoplasmic reticulum (ER) stress and induces selective cell death in multiple cell line models (in vitro). MATERIALS AND METHODS: We assessed the effects of the combination of these drugs on human NSCLC cell lines in vitro using cell proliferation assay and performed preclinical treatment studies using cell line-derived xenograft mouse models in vivo. RESULTS: In vitro, this combination enhanced inhibition of NSCLC cell proliferation with increased proteotoxicity, including ER stress, and apoptosis. In vivo, the growth of human NSCLC xenograft tumors was inhibited, which correlated with increased apoptosis and induction of ER stress as well as NSCLC growth in vitro. CONCLUSION: Our findings suggest that the induction of proteotoxicity provides a promising new target for developing anticancer drugs.
Subject(s)
Lung Neoplasms , Nelfinavir , Apoptosis , Cell Line, Tumor , Cell Proliferation , Chloroquine/pharmacology , Heterografts , Humans , Lung Neoplasms/drug therapy , Nelfinavir/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND/AIM: Small-cell lung cancer (SCLC) is aggressive and confers poor prognosis. Although SCLC shows more response to chemotherapy than other types of lung cancer, it is difficult to cure because of its frequent recurrence. New drugs and molecular targets need to be identified. MATERIALS AND METHODS: We investigated the effect of nelfinavir, an HIV protease inhibitor, on SCLC cells and in preclinical treatment studies using SCLC patient-derived xenograft (PDX) mouse models. RESULTS: Nelfinavir inhibited SCLC cell proliferation and induced cell death in vitro, which was caused by induction of the unfolded protein response (UPR), inhibition of mammalian/mechanistic target of rapamycin (mTOR) activation, and reduction in the expression of SCLC-related molecules such as achaete-scute homolog 1 (ASCL1). In vivo, nelfinavir inhibited the growth of SCLC PDX tumors, which correlated with the induction of UPR and reduced expression of ASCL1. CONCLUSION: Nelfinavir is highly effective in SCLC in vitro and in vivo, suggesting possible incorporation of nelfinavir into clinical trials for patients with SCLC.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Repositioning , Nelfinavir/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Biomarkers , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Nelfinavir/therapeutic use , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/metabolism , Small Cell Lung Carcinoma/pathology , TOR Serine-Threonine Kinases/metabolism , Unfolded Protein Response/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Diet modifies the risk of colorectal cancer (CRC), and inconclusive evidence suggests that yogurt may protect against CRC. We analysed the data collected from two separate colonoscopy-based case-control studies. The Tennessee Colorectal Polyp Study (TCPS) and Johns Hopkins Biofilm Study included 5446 and 1061 participants, respectively, diagnosed with hyperplastic polyp (HP), sessile serrated polyp, adenomatous polyp (AP) or without any polyps. Multinomial logistic regression models were used to derive OR and 95 % CI to evaluate comparisons between cases and polyp-free controls and case-case comparisons between different polyp types. We evaluated the association between frequency of yogurt intake and probiotic use with the diagnosis of colorectal polyps. In the TCPS, daily yogurt intake v. no/rare intake was associated with decreased odds of HP (OR 0·54; 95 % CI 0·31, 0·95) and weekly yogurt intake was associated with decreased odds of AP among women (OR 0·73; 95 % CI 0·55, 0·98). In the Biofilm Study, both weekly yogurt intake and probiotic use were associated with a non-significant reduction in odds of overall AP (OR 0·75; 95 % CI 0·54, 1·04) and (OR 0·72; 95 % CI 0·49, 1·06) in comparison with no use, respectively. In summary, yogurt intake may be associated with decreased odds of HP and AP and probiotic use may be associated with decreased odds of AP. Further prospective studies are needed to verify these associations.
Subject(s)
Colonic Polyps/epidemiology , Diet , Yogurt , Adenomatous Polyps/epidemiology , Adult , Aged , Case-Control Studies , Colonic Polyps/pathology , Colonoscopy , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/pathology , Female , Humans , Male , Middle Aged , Odds Ratio , Probiotics/administration & dosage , Risk Factors , Sex Factors , Tennessee/epidemiologyABSTRACT
The microbiome is increasingly recognized for its role in multiple aspects of cancer development and treatment, specifically in response to checkpoint inhibitors. While checkpoint inhibitors have revolutionized cancer treatment by producing durable anti-tumor responses, only a minority of patients respond to the available immunotherapy drugs and accurate, sensitive and specific microbiome predictors of response to treatment remain elusive. Additionally, the specific mechanisms linking the microbiome and host immunological responses remain unclear. In this review, we examine the evidence for the gut microbiome's association with anti-tumor responses to checkpoint inhibitors in the treatment of melanoma, non-small cell lung cancer, and renal cell carcinoma. Furthermore, we discuss the current evidence available from murine models seeking to explain the immunological mechanisms that may drive this process. While this work is promising in defining the impact of gut microbiota in cancer treatment, many unanswered questions indicate the need for additional human and experimental studies.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Melanoma/drug therapy , Microbiota , Animals , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/etiology , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Gastrointestinal Microbiome/genetics , Gastrointestinal Microbiome/immunology , Humans , Immunomodulation/drug effects , Lung Neoplasms/etiology , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Melanoma/etiology , Microbiota/drug effects , Microbiota/immunology , Molecular Targeted Therapy , Prognosis , Treatment OutcomeABSTRACT
Poly (ADP-ribose) polymerase inhibitors (PARPi) have been developed and tested in a context of combining it with double-stranded (ds) DNA repair defects or inhibitors, as PARP inhibitor impairs single-stranded (ss) DNA break repair, resulting in the activation of the dsDNA break repair machinery. Rapamycin has been widely prescribed for more than a decade and recent studies have revealed that it may inhibit dsDNA break repair. The combination of the PARP inhibitor olaparib and rapamycin synergistically inhibited cell proliferation in non-small cell lung cancer (NSCLC) cells, and even in triple negative breast cancer (TNBC) cells with BRCA1 mutations. Rad51, which forms a polymer on ssDNA upon dsDNA breaks, plays an essential role in homologous recombination. Olaparib induced Rad51 focus formation, while rapamycin successfully inhibited it both in vivo and in vitro, suggesting that this combination worked through the blocking of both ssDNA break repair and dsDNA break repair; hence the cells cannot go through the G2/M checkpoint. The protein level of PARP was a predictive marker for both PAR activity and Rad51 focus formation in this combination. Collectively, these data suggest that this combination could have therapeutic potential in the treatment of cancer with high PARP expression, or in combination with cytotoxic chemotherapy or radiotherapy.
ABSTRACT
Alterations in EGFR, KRAS, and ALK are oncogenic drivers in lung cancer, but how oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. Immunosuppression likely contributes to lung cancer, because drugs that inhibit immune checkpoints like PD-1 and PD-L1 have clinical benefit. Here, we show that activation of the AKT-mTOR pathway tightly regulates PD-L1 expression in vitro and in vivo. Both oncogenic and IFNγ-mediated induction of PD-L1 was dependent on mTOR. In human lung adenocarcinomas and squamous cell carcinomas, membranous expression of PD-L1 was significantly associated with mTOR activation. These data suggest that oncogenic activation of the AKT-mTOR pathway promotes immune escape by driving expression of PD-L1, which was confirmed in syngeneic and genetically engineered mouse models of lung cancer where an mTOR inhibitor combined with a PD-1 antibody decreased tumor growth, increased tumor-infiltrating T cells, and decreased regulatory T cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Oncogene Protein v-akt/metabolism , Programmed Cell Death 1 Receptor/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Humans , Lung Neoplasms/pathology , Mice , TransfectionABSTRACT
Nelfinavir is an HIV protease inhibitor being repurposed as an anti-cancer agent in preclinical models and in small oncology trials, yet the MTD of nelfinavir has not been determined. Therefore, we conducted a Phase Ia study to establish the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of nelfinavir in subjects with advanced solid tumors. Adults with refractory cancers were given oral nelfinavir twice daily with pharmacokinetic and pharmacodynamic analyses. Twenty-eight subjects were enrolled. Nelfinavir was generally well tolerated. Common adverse events included diarrhea, anemia, and lymphopenia, which were mostly mild. The DLT was rapid-onset neutropenia that was reversible. The MTD was established at 3125 mg twice daily. In an expansion cohort at the MTD, one of 11 (9%) evaluable subjects had a confirmed partial response. This, plus two minor responses, occurred in subjects with neuroendocrine tumors of the midgut or pancreatic origin. Thirty-six percent of subjects had stable disease for more than 6 months. In peripheral blood mononuclear cells, Nelfinavir inhibited AKT and induced markers of ER stress. In summary, nelfinavir is well tolerated in cancer patients at doses 2.5 times the FDA-approved dose for HIV management and showed preliminary activity in tumors of neuroendocrine origin.
Subject(s)
Antineoplastic Agents/therapeutic use , HIV Protease Inhibitors/therapeutic use , Nelfinavir/therapeutic use , Neoplasms/drug therapy , Administration, Oral , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Drug Administration Schedule , Female , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/adverse effects , HIV Protease Inhibitors/pharmacokinetics , Humans , Male , Maryland , Maximum Tolerated Dose , Middle Aged , Nelfinavir/administration & dosage , Nelfinavir/adverse effects , Nelfinavir/pharmacokinetics , Neoplasms/pathology , Time Factors , Tomography, X-Ray Computed , Treatment Outcome , Young AdultABSTRACT
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-EGFR(L858R/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 PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/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, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.
Subject(s)
Adenocarcinoma/metabolism , ErbB Receptors/genetics , Lung Neoplasms/metabolism , SOXB1 Transcription Factors/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Erlotinib Hydrochloride , Gene Expression , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice, Transgenic , Mutation , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Quinazolines/pharmacology , SOXB1 Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Glutamates/pharmacology , Guanine/analogs & derivatives , Lung Neoplasms/drug therapy , Sirolimus/pharmacology , Thymidylate Synthase/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation , Drug Synergism , Female , Glutamates/administration & dosage , Guanine/administration & dosage , Guanine/pharmacology , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Male , Mice , Mice, Nude , Pemetrexed , Xenograft Model Antitumor AssaysABSTRACT
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-I and insulin. Here, we used liver IGF-I-deficient (LID) mice to determine the importance of IGF-I in NNK-induced lung tumorigenesis and chemoprevention by metformin. LID mice had decreased lung tumor multiplicity and burden compared with wild-type (WT) mice. Metformin further decreased lung tumorigenesis in LID mice without affecting IGF-I levels, suggesting that metformin can act through IGF-I-independent mechanisms. In lung tissues, metformin decreased phosphorylation of multiple receptor tyrosine kinases (RTK) 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 [(14)C]-metformin showed that uptake of metformin was high in liver but four-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.
Subject(s)
AMP-Activated Protein Kinases/physiology , Cell Transformation, Neoplastic/drug effects , Hypoglycemic Agents/pharmacology , Insulin-Like Growth Factor I/physiology , Lung Neoplasms/prevention & control , Metformin/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Carcinogens/toxicity , Cell Transformation, Neoplastic/pathology , Energy Metabolism/drug effects , Enzyme-Linked Immunosorbent Assay , Female , Hypoglycemic Agents/pharmacokinetics , Insulin-Like Growth Factor I/antagonists & inhibitors , Lung Neoplasms/chemically induced , Male , Metformin/pharmacokinetics , Mice , Mice, Inbred A , Mice, Knockout , Nitrosamines/toxicity , Phosphorylation/drug effects , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Tissue DistributionABSTRACT
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.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Neoplasms/drug therapy , Neoplasms/prevention & control , Animals , Clinical Trials as Topic , Disease Models, Animal , HumansABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Phosphatidylinositol Phosphates/chemistry , Phosphatidylinositol Phosphates/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence AlignmentABSTRACT
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.
Subject(s)
CDC2 Protein Kinase/analysis , Drug Resistance, Neoplasm/genetics , Lung Neoplasms/genetics , CDC2 Protein Kinase/genetics , Cell Line, Tumor , Cytoplasm/chemistry , Gene Expression Profiling , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/mortality , Predictive Value of Tests , Prognosis , Survival RateABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung , Chromones/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Lipids/pharmacology , Morpholines/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Chromones/chemistry , Cluster Analysis , Enzyme Inhibitors/pharmacology , Gene Expression Profiling , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Morpholines/chemistry , Phosphatidylinositols/chemistry , Phosphatidylinositols/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Reproducibility of ResultsABSTRACT
Myoinositol is an isomer of glucose that has chemopreventive activity in animal models of cancer. In a recent phase I clinical trial, myoinositol administration correlated with a statistically significant regression of preexisting bronchial dysplastic lesions in heavy smokers. To shed light on the potential mechanisms involved, activation of Akt and extracellular signal-regulated kinase (ERK), two kinases that control cellular proliferation and survival, was assessed in 206 paired bronchial biopsies from 21 patients who participated in this clinical trial. Before myoinositol treatment, strongly positive staining for activation of Akt was detected in 27% of hyperplastic/metaplastic lesions and 58% of dysplastic lesions (P = 0.05, chi(2) test). There was also a trend toward increased activation of ERK (28% in regions of hyperplasia/metaplasia to 42% of dysplastic lesions). Following myoinositol treatment, significant decreases in Akt and ERK phosphorylation were observed in dysplastic (P < 0.01 and 0.05, respectively) but not hyperplastic/metaplastic lesions (P > 0.05). In vitro, myoinositol decreased endogenous and tobacco carcinogen-induced activation of Akt and ERK in immortalized human bronchial epithelial cells, which decreased cell proliferation and induced a G(1)-S cell cycle arrest. These results show that the phenotypic progression of premalignant bronchial lesions from smokers correlates with increased activation of Akt and ERK and that these kinases are targets of myoinositol. Moreover, they suggest that myoinositol might cause regression of bronchial dysplastic lesions through inhibition of active Akt and ERK.