LKB1 Inactivation Research Articles

TET2-STAT3-CXCL5 nexus promotes neutrophil lipid transfer to fuel lung adeno-to-squamous transition.

Phenotypic plasticity is a rising cancer hallmark, and lung adeno-to-squamous transition (AST) triggered by LKB1 inactivation is significantly associated with drug resistance. Mechanistic insights into AST are urgently needed to identify therapeutic vulnerability in LKB1-deficient lung cancer. Here, we find that ten-eleven translocation (TET)-mediated DNA demethylation is elevated during AST in KrasLSL-G12D/+; Lkb1L/L (KL) mice, and knockout of individual Tet genes reveals that Tet2 is required for squamous transition. TET2 promotes neutrophil infiltration through STAT3-mediated CXCL5 expression. Targeting the STAT3-CXCL5 nexus effectively inhibits squamous transition through reducing neutrophil infiltration. Interestingly, tumor-infiltrating neutrophils are laden with triglycerides and can transfer the lipid to tumor cells to promote cell proliferation and squamous transition. Pharmacological inhibition of macropinocytosis dramatically inhibits neutrophil-to-cancer cell lipid transfer and blocks squamous transition. These data uncover an epigenetic mechanism orchestrating phenotypic plasticity through regulating immune microenvironment and metabolic communication, and identify therapeutic strategies to inhibit AST.

Abstract 1653: Heme sequestration modulates the tumor immune microenvironment in KRAS mutation/LKB1 inactivation non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. KRAS mutations and LKB1 inactivation commonly occur in NSCLC and are associated with poor prognosis. Several studies enunciate the role of the immune system in lung cancer progression through cancer cells' ability to escape immune surveillance. Therefore, understanding NSCLC progression requires the examination of the molecular mechanisms behind this process. The importance of oxidative phosphorylation (OXPHOS) and mitochondrial metabolism in lung cancer has been continuously emphasized; OXPHOS and mitochondrial biogenesis markers predict low survival in NSCLC patients. Additionally, heme is an essential molecule in the OXPHOS pathway, and previous studies have shown that heme sequestration using heme sequestering protein 2 (HeSP2) arrests tumor progression. HeSP2 can potentially normalize the tumor immune microenvironment (TIME) via multiple mechanisms including the normalization of vessels which may promote immune cell infiltration, the reduction of hypoxia which may promote antitumor immune function, and the reduction of heme levels which potentially decreases the production of immunosuppressive enzymes requiring heme as a cofactor. In this study, control and HeSP2-treated tumors generated from a genetically engineered mouse model LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc) infected with AdenoCre virus and sacrificed at 10 months were used for immunohistochemical analysis. We examined immune markers under HeSP2 treatment to determine the immune system response to heme targeting and its effects on tumor progression. Results demonstrated that HeSP2 can modulate the tumor immune microenvironment increasing the levels of immune markers, which suggests that heme targeting can potentiate immunotherapy efficacy in lung cancer treatment. Citation Format: Maria del Carmen Chacon Castro, Eranda Berisha, Narges Salamat, Li Zhang. Heme sequestration modulates the tumor immune microenvironment in KRAS mutation/LKB1 inactivation non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1653.

LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer

LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPα, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation.

Abstract 2937: Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth

Abstract The goal of this study is to define the role of NEDD9 in formation of progression of non-small cell lung cancer (NSCLC). NSCLC has a low survival rate, with metastasis contributing to the vast majority of deaths. NEDD9 expression has been shown to be repressed by LKB1, and upregulated in LKB1-mutated NSCLC; in these tumors, elevated NEDD9 promotes metastasis, serving as a scaffolding intermediate in the integrin and receptor tyrosine kinase signaling cascades, promoting pro-invasive signaling. In this study, we for the first time assessed the consequences for cancer formation after introduction of a constitutive null allele of Nedd9 (KPN mice) in the Krastm4Tyj/J/Trp53tm1Brn/J (KP mice) Adeno-cre inducible murine model of NSCLC, in the context of intact Lkb1. Unexpectedly, KPN mice presented with significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Orthotopic re-implantation of KP and KPN tumor into syngeneic mice with wt and null Nedd9 genotypes demonstrated that the effect of Nedd9 loss on promoting tumor growth is cell-autonomous. Proteomic analysis of KPN versus KP tumors revealed that more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative, pro-invasive Nedd9 partner proteins Src and Fak. Treatment of mice with the Src inhibitor dasatinib significantly reduced, but did not eliminate, tumor growth in KP and KPN mice. As an alternative hypothesis, we considered that Ras-dependent NSCLC tumors require active autophagy, a process dependent on activation of the kinases Lkb1 and Ampk; given Lkb1 represses Nedd9, we investigated whether Nedd9 reciprocally regulates Lkb1. We found that KPN tumors specifically and significantly upregulated active Lkb1 based on a mechanism of post-transcriptional activation, causing increased expression of the Lkb1 substrate Ampk and downstream effectors of autophagy, including elevated appearance of LC3-positive autophagosomes in KPN NSCLC tumor tissue. NEDD9 depletion in human NSCLC cell lines similarly elevated LKB1 activation. In vivo treatment of KPN and KP mice with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors, emphasizing the importance of autophagy for the growth differential. In sum, these data for the first time identify and define a previously unknown role for the pro-metastatic protein NEDD9 in control of autophagy, based on a role as a negative regulator of the Lkb1-Ampk signaling axis in NSCLC. Citation Format: Alexander Y. Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Anna Kiseleva, Douglas Flieder, Ilya Serebriiskii, Harvey Hensley, Erica Golemis. Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2937.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Leflunomide Suppresses the Growth of LKB1-Inactivated Tumors in the Immune-Competent Host and Attenuates Distant Cancer Metastasis.

Liver kinase B1 (LKB1)-inactivated tumors are vulnerable to the disruption of pyrimidine metabolism, and leflunomide emerges as a therapeutic candidate because its active metabolite, A77-1726, inhibits dihydroorotate dehydrogenase, which is essential for de novo pyrimidine biosynthesis. However, it is unclear whether leflunomide inhibits LKB1-inactivated tumors in vivo, and whether its inhibitory effect on the immune system will promote tumor growth. Here, we carried out a comprehensive analysis of leflunomide treatment in various LKB1-inactivated murine xenografts, patient-derived xenografts, and genetically engineered mouse models. We also generated a mouse tumor-derived cancer cell line, WRJ388, that could metastasize to the lung within a month after subcutaneous implantation in all animals. This model was used to assess the ability of leflunomide to control distant metastasis. Leflunomide treatment shrank a HeLa xenograft and attenuated the growth of an H460 xenograft, a patient-derived xenograft, and lung adenocarcinoma in the immune-competent genetically engineered mouse models. Interestingly, leflunomide suppressed tumor growth through at least three different mechanisms. It caused apoptosis in HeLa cells, induced G1 cell-cycle arrest in H460 cells, and promoted S-phase cell-cycle arrest in WRJ388 cells. Finally, leflunomide treatment prevented lung metastasis in 78% of the animals in our novel lung cancer metastasis model. In combination, these results demonstrated that leflunomide utilizes different pathways to suppress the growth of LKB1-inactivated tumors, and it also prevents cancer metastasis at distant sites. Therefore, leflunomide should be evaluated as a therapeutic agent for tumors with LKB1 inactivation.

Abstract 392: LKB-inactivated lung cancer cells exhibit dependency on INSL4 signaling

Abstract Lung cancer is the leading cause of cancer deaths worldwide. The LKB1 (STK11) tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Currently, no effective treatments are available for LKB1-inactivated lung cancer. Elucidation of critical signaling downstream of LKB1-inactivation has the potential to uncover rational therapeutic targets for this unique and prevalent molecular subset of lung cancers. In this study, we identified INSL4 (insulin-like 4), a member of the insulin/IGF/relaxin superfamily, as a novel downstream target of LKB1-deficiency through gene expression profiling analysis. We observed that INSL4 expression was highly induced through aberrant CRTC-CREB activation subsequent to LKB1 inactivation. INSL4 was highly expressed in human LKB1-deficient NSCLC cells and primary tumors while undetectable in all normal tissues except in the placenta. High INSL4 expression was associated with more advanced lung cancer and worse patient survival. INSL4 depletion inhibited the growth and viability of LKB1-null NSCLC cells in vitro and blocked the growth of lung cancer xenografts. Moreover, expression profiling identified an INSL4-regulated gene program that is associated with the regulation of cell cycle, growth, and survival. In conclusion, LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers. Citation Format: Rongqiang Yang, Steven W. Li, Zirong Chen, Xin Zhou, Wei Ni, Dongtao A. Fu, Jianrong Lu, Frederic J. Kaye, Lizi Wu. LKB-inactivated lung cancer cells exhibit dependency on INSL4 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 392.

Kras and Lkb1 mutations synergistically induce intraductal papillary mucinous neoplasm derived from pancreatic duct cells

ObjectivePancreatic cancer can arise from precursor lesions called intraductal papillary mucinous neoplasms (IPMN), which are characterised by cysts containing papillae and mucus-producing cells. The high frequency of KRAS mutations in...

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation.

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was “missed” due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Genome editing of LKB1 (STK11) mutation in A549 lung cancer cells using CRISPR/Cas9 system

Mutations and deletions of the tumor suppressor LKB1 (STK11) are common in several malignancies, especially in non-small lung cancer cells (NSCLC). In that cell type, LKB1 inactivation coexists with mutations at other important cancer genes, including KRAS and TP53. The mutant LKB1 indirectly increases transcription of genes involved in metastasis, including COX-2. LKB1 directly activates AMPK decreasing mTOR activity and plays roles in several other processes such as cell metabolism, cell polarity, apoptosis and DNA damage via p53 interaction. Metformin, an antidiabetic drug, have shown antitumor effects, sensitizes cells in coadjuvant cisplatin treatment and acts in AMPK independent and dependent mechanisms. Thus, we propose the application of CRISPR/Cas9 system as genome editing tool to correct the LKB1 nonsense mutation and restore protein expression, providing new insights regarding cisplatin resistance, survival and metformin response in A549 lung cancer cells.

Role of INSL4 Signaling in Sustaining the Growth and Viability of LKB1-Inactivated Lung Cancer.

The LKB1 tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Targeted therapies for LKB1-inactivated lung cancer are currently unavailable. Identification of critical signaling components downstream of LKB1 inactivation has the potential to uncover rational therapeutic targets. Here we investigated the role of INSL4, a member of the insulin/IGF/relaxin superfamily, in LKB1-inactivated NSCLCs. INSL4 expression was analyzed using global transcriptome profiling, quantitative reverse transcription PCR, western blotting, enzyme-linked immunosorbent assay, and RNA in situ hybridization in human NSCLC cell lines and tumor specimens. INSL4 gene expression and clinical data from The Cancer Genome Atlas lung adenocarcinomas (n = 515) were analyzed using log-rank and Fisher exact tests. INSL4 functions were studied using short hairpin RNA (shRNA) knockdown, overexpression, transcriptome profiling, cell growth, and survival assays in vitro and in vivo. All statistical tests were two-sided. INSL4 was identified as a novel downstream target of LKB1 deficiency and its expression was induced through aberrant CRTC-CREB activation. INSL4 was highly induced in LKB1-deficient NSCLC cells (up to 543-fold) and 9 of 41 primary tumors, although undetectable in all normal tissues except the placenta. Lung adenocarcinomas from The Cancer Genome Atlas with high and low INSL4 expression (with the top 10th percentile as cutoff) showed statistically significant differences for advanced tumor stage (P < .001), lymph node metastasis (P = .001), and tumor size (P = .01). The INSL4-high group showed worse survival than the INSL4-low group (P < .001). Sustained INSL4 expression was required for the growth and viability of LKB1-inactivated NSCLC cells in vitro and in a mouse xenograft model (n = 5 mice per group). Expression profiling revealed INSL4 as a critical regulator of cell cycle, growth, and survival. LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.

Abstract LB-085: A new role for LKB1 to regulate Heat Shock Protein 90 activity

Abstract Approximately 30% of human non-small cell lung cancer (NSCLC) patients harbor a somatic KRAS mutation resulting, in aberrant activation of downstream signaling pathways that control cell proliferation, cell growth, and cell survival. Importantly, alleles of LKB1, a serine/threonine kinase that functions as a tumor suppressor, are somatically inactivated in ~30% of NSCLCs within KRAS-mutant NSCLC. The loss of LKB1 gives rise to aggressive, highly metastatic, and highly drug resistant tumors. We have previously demonstrated that the inactivation of the tumor suppressor lkb1 rendered mutant kras murine NSCLC resistant to targeted agents including BET bromodomain and kinase inhibitors. However, the mechanism by which LKB1 inactivation attenuates the efficacy of the therapies remains elusive. Consequently, investigation of the molecular-basis of drug resistance to formulate effective therapies for this KRAS-mutated and LKB1-inactivated (KRAS/LKB1) subset of patients is warranted. Using lung adenocarcinoma cells that lack LKB1 expression, we have established isogenic cells that express vector control, wild-type LKB1, or kinase-dead LKB1. Our preliminary data using these isogenic cell lines suggest the previously unrealized function of LKB1 to activate HSP90 to stabilize client signaling proteins. Furthermore, we have performed p23 immunoprecipitation (IP) followed by Western blot for HSP90 to assess the HSP90 ATPase activity. In the cells ectopically expressing wild-type LKB1, p23 co-immunoprecipitated with HSP90 showing that HSP90 is in active conformation. In contrast, few HSP90 co-immunoprecipitated with p23 in the cells that express the kinase-dead version of LKB1 or vector control, demonstrating that little HSP90 is in active conformation without LKB1 kinase activity. Using NetworKIN, a web-based bioinformatic analysis, we find putative LKB1 phosphorylation motifs on HSP90. These results suggest that LKB1 phosphorylates and activates HSP90. Consequently, the inactivation of LKB1 contributes to the destabilization of key HSP90 client signaling proteins and inadvertently promotes compensatory mechanisms to stabilize the select signaling molecules. Using our LKB1-isogenic cell lines, we demonstrated that LKB1 activates HSP90 to stabilize RAF1, resulting in MAPK pathway activation. In contrast, LKB1 inactivation led to reduced HSP90 activity, which reduced MAPK activity and elevated IGF-1R-mediated phosphatidylinositol 3-kinase (PI3-K) activity in vivo and in vitro models of mutant KRAS NSCLC. The aberrant activation of PI3-K pathway augments survival by the stabilization of the anti-apoptotic molecule, MCL1. The use of HSP90 inhibitors does not fully inhibit MAPK or PI3-K pathways due to the compensatory mechanisms chaperoning the IGF-1R axis. Importantly, we confirmed that canonical LKB1-AMPK-mTOR pathway does not fully control PI3K or MAPK signaling. Taken together, our data supports the presence of an HSP90-independent mechanism to stabilize kinases, including IGF-1R, to make KRAS/LKB1 NSCLC resistant to targeted therapies. Citation Format: Jeffrey H. Becker, Michael P. Kahle, Margaret Soucheray, Ines Pulido, Fatima Al-Shahrour, Manuel Sanchez del Pino, Kwok-Kin Wong, Julian Carretero, Takeshi Shimamura. A new role for LKB1 to regulate Heat Shock Protein 90 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-085.

Combination of metformin plus TKI vs. TKI alone in EGFR(+) LUNG adenocarcinoma: A randomized phase II study.

9013Background: Metformin has been shown to have antitumor activity by increasing AMPK through different mechanisms involving tumor suppressor gene, LKB1. LKB1 inactivation is common in Non-small c...

LKB1 loss cooperating with BRAF V600E promotes melanoma cell invasion and migration by up-regulation MMP-2 via PI3K/Akt/mTOR pathway.

The serine/threonine kinase LKB1, act as a tumor suppressor, has been reported in several sporadic cancers. However, how the loss of LKB1 promotes melanoma invasion and metastasis remains incompletely understood. In this study, we inactivated LKB1expression by RNA interference in BRAF mutation and wild type melanoma cells respectively. We found LKB1 inactivation cooperate with BRAF V600E lead to melanoma cells more aggressive by a series of experiments including wound scratch test, Transwell assay. While single alteration, either LKB1 loss or BRAF V600E, fails to enhance melanoma cells invasion ability. Mechanistically, LKB1 loss synergism with BRAF V600E resulted in the activation of the PI3K/Akt/mTOR signaling pathway and significant up-regulation expression of MMP-2. In addition, LKB1 expression in human melanoma tissues was negatively associated with MMP-2 expression in the presence of BRAF V600E. Thus, our findings indicate a probable explanation on LKB1 function as a tumor suppressor in melanoma and a new therapeutic strategy for melanoma by targeting on BRAF and LKB1 together.

Abstract 2775: CPS1 as a therapeutic target and prognostic indicator in LKB1-inactivated lung adenocarcinoma

Abstract Liver Kinase B1 (LKB1), encoded by STK11, is a tumor suppressor and somatically mutated in approximately 20% of lung adenocarcinoma. Aside from the effects of LKB1 inactivation on tumor initiation, LKB1-mutant cancers are biologically distinct from cancer with functional LKB1, and the loss of LKB1 uniquely confers invasive and metastatic properties in genetically engineered mouse models of cancer. While various pathways, including energy metabolism, cell polarity, and cell growth, are regulated by LKB1 and can play a pleiotropic role in cancer initiation and progression, no therapies are currently available for clinical use that specifically target LKB1 inactivation. Therefore, elucidation of the functional mechanisms associated with LKB1 inactivation has translational relevance. We analyzed proteomic profiles of 45 lung adenocarcinoma cell lines with and without LKB1 inactivation to identify molecular features associated with LKB1 inactivation. Carbamoyl phosphate synthase 1 (CPS1) was identified as a markedly overexpressed protein in LKB1-inactivated lung adenocarcinoma cell lines. CPS1 is the first rate-limiting mitochondrial enzyme in the urea cycle, and plays an intricate role in arginine and pyrimidine metabolism. CPS1 knockdown reduced cell growth, decreased levels of metabolites associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with conventional chemotherapy agents including gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis using 305 lung adenocarcinoma tumors revealed that CPS1 was expressed in 65.7% of LKB1-negative and only 5.0% of LKB1-positive lung adenocarcinomas. In addition, CPS1 expression was significantly and independently associated with poor overall survival of lung adenocarcinoma patients (P &amp;lt; 0.0001, HR = 3.03, 95% CI 1.74-5.25). Concordant with the results of tissue microarray, analysis of the Cancer Genome Atlas (TCGA) dataset consisting 403 lung adenocarcinomas revealed that high CPS1 mRNA expression was significantly associated with worse overall survival and is an independent prognostic indicator of lung adenocarcinoma (P = 0.005, HR = 2.31, 95% CI 1.28-4.16). Our findings suggest functional relevance of CPS1 and its association with worse outcome in LKB1-inactivated lung adenocarcinoma. Therefore, CPS1 is a promising therapeutic target in combination with other chemotherapy agents, as well as a prognostic biomarker, enabling us to personalize the treatment of LKB1-inactivated lung adenocarcinoma. Citation Format: Muge Celiktas, Ichidai Tanaka, Satyendra Chandra Tripathi, Johannes F. Fahrmann, Clemente Aguilar-Bonavides, Pamela Villalobos, Oliver Delgado, Dilsher Dhillon, Jennifer B. Dennison, Edwin J. Ostrin, Hong Wang, Carmen Behrens, Kim-Anh Do, Adi F. Gazdar, Samir M. Hanash, Ayumu Taguchi. CPS1 as a therapeutic target and prognostic indicator in LKB1-inactivated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2775. doi:10.1158/1538-7445.AM2017-2775

Abstract 2907: Extracellular matrix remodeling triggered by lysyl oxidase inhibition promote the lung adenocarcinoma to squamous cell carcinoma transition independent of LKB1 status

Abstract Introduction: Lung adeno-squamous cell carcinoma represents as the most malignant subtype of non-small cell lung cancer. We have previously shown that Lkb1-deficiency triggered the lung adenocarcinoma (ADC) to squamous cell carcinoma (SCC) transition (AST) in mice through extracellular matrix remodeling, in which lysyl oxidase (LOX) plays an important role. Considering the high frequency of P53 mutations in human lung cancer, we reason it's attractive to test if LOX inhibition could modulate the lung cancer plasticity and subtype transition independent of LKB1 status.Methods: KrasG12D/P53L/L (KP) mice were used to treat with BAPN or DPA,two LOX inhibitors, daily via intraperitoneal injection post 4 weeks of Adeno-Cre infection for 6-8 weeks and then harvested the lungs for pathological analyses. The histology and expression of related markers were analyzed by H&amp;E, IHC staining, quantitative PCR and western blot. Results: BAPN/DPA treatment could result in a dramatic reduction of collagen deposition. We found no significant changes of total tumor number, but decreased average tumor size and tumor burden in treatment groups. Detailed pathological analyses showed that a few tumors from treatment group displayed typical squamous pathology, with the expression of squamous markers P63 and K14. In contrast, the tumors from control groups were adenomatous pathology and only expressing ADC markers TTF1 and SP-C. We further found that either BAPN or DPA treatment in KP mice promoted apoptosis and inhibited cell proliferation in ADC. Interestingly, we found the transited SCC showed almost no growth arrest and low apoptosis rate after BAPN or DPA treatment, indicative of the resistance to LOX inhibitors. ECM deprivation is a major cause of oxidative stress that contributes to the accumulation of reactive oxygen species (ROS) through the deregulation of certain metabolic pathways. We then checked the level of 8-hydroxydeoxyguanosine (8-oxo-dGuo), a marker of DNA oxidative modification by ROS. In treatment groups, 8-oxo-dGuo was significantly lower in SCC than ADC. In our previous work done in KL model, we found that the ROS level was significantly up-regulated by LKB1 inactivation, which functionally modulated the AST. However, in KP mouse model, either LKB1 or p-AMPK level showed no significant change with or without BAPN/DPA treatment.Conclusions: These data demonstrated that pharmacological LOX inhibition could trigger the ADC to SCC transition independent of LKB1 status, highlight a non-cancer-cell autonomous role in phenotypic transition. Moreover, this lineage transition confers lung cancer with strong survival capability under environmental stress as well as the acquisition of drug resistance. Citation Format: Shun Yao, Hongbin Ji. Extracellular matrix remodeling triggered by lysyl oxidase inhibition promote the lung adenocarcinoma to squamous cell carcinoma transition independent of LKB1 status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2907. doi:10.1158/1538-7445.AM2017-2907

Lkb1 deletion in murine B lymphocytes promotes cell death and cancer.

LKB1 (also known as STK11) is a potent tumor suppressor in solid tumors, such as melanoma and lung adenocarcinoma, but inactivation in hematopoietic cells causes cell death without signs of tumorigenesis. We noted somatic LKB1 deletion or mutation at low frequency in human B-cell lymphoma. To determine if LKB1 inactivation is a passenger or driver event in lymphoid cancers, we examined the effects of conditional inactivation of Lkb1 in murine lymphocytes. Consistent with prior reports, Lkb1 deletion in either T or B cells resulted in massive, lineage-specific apoptosis. Surprisingly, despite an 80% reduction of peripheral B-cell number, animals harboring somatic B-lineage Lkb1 deletion developed aggressive B-cell lymphoma with high penetrance and moderate latency. Malignant B cells exhibited somatic Lkb1 recombination. In contrast, Lkb1 deletion in T cells did not promote tumorigenesis. Concomitant Ras activation with Lkb1 deletion reduced T-cell apoptosis, but did not enhance tumor formation in T or B cells. These results suggest that although physiologic LKB1 expression exerts a potent pro-survival effect in lymphocytes, LKB1 inactivation nonetheless facilitates transformation of B, but not T, lymphocytes.

Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2

Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours.

Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2

Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2