Tumor Suppressor Liver Kinase B1 Research Articles

Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1)‐deficient lung tumor growth

Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non‐small cell lung cancer (NSCLC), autophagy supports Kras‐driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20–30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1‐deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7−/− tumors compared to mice bearing wild‐type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1‐deficient Kras‐driven lung tumor growth, tumor‐derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation‐induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation‐induced Atg7 null TDCLs death, indicating that autophagy is required to maintain the free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy‐mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase‐1 (CPT‐1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1‐deficient lung tumor.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award and start‐up funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LKB1 pro-oncogenic activity triggers cell survival in circulating tumor cells.

During intravasation, circulating tumor cells (CTCs) detach from the epithelium of origin and begin the epithelial‐to‐mesenchymal transition (EMT) process, where they lose epithelial features and pass through the endothelium to enter circulation. Although detachment from the extracellular matrix is a strong source of metabolic stress, which induces anoikis, CTCs can survive. Recently, the tumor suppressor liver kinase B1 (LKB1) has gained attention for its role as a proto‐oncogene in restoring the correct ATP/AMP ratio during metabolic stress. The aim of this study was to assess LKB1 expression in epithelial‐negative CTCs isolated from patients with metastatic breast cancer and to characterize its possible association with EMT and stemness features. Transcriptome analysis of EpCAM‐negative CTCs indicated that over 25% of patients showed enhanced LKB1 levels, while almost 20% of patients showed enhanced levels of an EMT transcription factor known as ZEB1. Transcriptome and immunofluorescence analyses showed that patients with enhanced LKB1 were correspondingly ZEB1 negative, suggesting complementary activity for the two proteins. Only ZEB1 was significantly associated with cancer stem cell (CSC) markers. Neither LKB1 nor ZEB1 upregulation showed a correlation with clinical outcome, while enhanced levels of stemness‐associated CD44 correlated with a lower progression‐free and overall survival. Ex vivo models showed that MDA‐MB‐231, a mesenchymal tumor cell line, grew in suspension only if LKB1 was upregulated, but the MCF‐7 epithelial cell line lost its ability to generate spheroids and colonies when LKB1 was inhibited, supporting the idea that LKB1 might be necessary for CTCs to overcome the absence of the extracellular matrix during the early phases of intravasation. If these preliminary results are confirmed, LKB1 will become a novel therapeutic target for eradicating metastasis‐initiating CTCs from patients with primary breast cancer.

Peripheral Neuropathy and Hindlimb Paralysis in a Mouse Model of Adipocyte-Specific Knockout of Lkb1

Brown adipose tissues (BAT) burn lipids to generate heat through uncoupled respiration, thus representing a powerful target to counteract lipid accumulation and obesity. The tumor suppressor liver kinase b1 (Lkb1) is a key regulator of cellular energy metabolism; and adipocyte-specific knockout of Lkb1 (Ad-Lkb1 KO) leads to the expansion of BAT, improvements in systemic metabolism and resistance to obesity in young mice. Here we report the unexpected finding that the Ad-Lkb1 KO mice develop hindlimb paralysis at mid-age. Gene expression analyses indicate that Lkb1 KO upregulates the expression of inflammatory cytokines in interscapular BAT and epineurial brown adipocytes surrounding the sciatic nerve. This is followed by peripheral neuropathy characterized by infiltration of macrophages into the sciatic nerve, axon degeneration, reduced nerve conductance, and hindlimb paralysis. Mechanistically, Lkb1 KO reduces AMPK phosphorylation and amplifies mammalian target-of-rapamycin (mTOR)-dependent inflammatory signaling specifically in BAT but not WAT. Importantly, pharmacological or genetic inhibition of mTOR ameliorates inflammation and prevents paralysis. These results demonstrate that BAT inflammation is linked to peripheral neuropathy.

Abstract 5417: The tumor suppressor klotho: A master regulator of metabolism in breast cancer

Abstract One of the hallmarks of cancer is reprogramming of energy metabolism, characterized by a shift to aerobic glycolysis (Warburg effect). A major regulator of this phenomenon is the IGF-1/Pi3K/AKT pathway. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. Klotho is a potent tumor suppressor in BC. As klotho is a potent inhibitor of the IGF-1/Pi3K/AKT pathway in BC, we hypothesized that it may revert the metabolic switch in BC cells. We first analyzed the effect of klotho on the energy sensor enzyme AMP-activated kinase (AMPK) and its down-stream effector acetyl CoA carboxylase (ACC). Overexpression of klotho, or treatment with the soluble protein, elevated AMPK and ACC phosphorylation in three BC cell lines. AMPK is activated by the tumor suppressor liver kinase B1 (LKB1) and elevated AMP/ATP ratio. Expression of a dominant negative LKB1 prevented activation of AMPK by klotho and decreased the ability of klotho to inhibit cell growth and migration, implying that klotho tumor suppressor activities are LKB1 dependent. We next analyzed the effects of klotho on critical components of glucose metabolism. Treatment with soluble klotho reduced expression of the glucose transporter GLUT1 and the key glycolytic enzymes hexokinase2 (HK2), phosphofructokinase1 (PFK1), pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase kinase 1(PDK1). Using the Seahorse analyzer we noted reduced oxidative mitochondrial metabolism and glycolysis following klotho treatment. Energy is obtained essentially in the mitochondria with the transfer of protons across the inner membrane that produces ATP. We, therefore, analyzed klotho effect on the mitochondria membrane potential and noted reduction in the mitochondria potential following treatment with klotho. Finally, we examined the effects of klotho on specific metabolites and also conducted an NMR-based metabolic profiling. In accordance with the alterations in signaling pathways and levels of glycolytic enzymes, klotho reduced glucose uptake and inhibited lactate, pyruvate and 3-hydroxy-butyrate production. Furthermore, klotho also inhibited ATP production. Taken together, our data indicate klotho as a regulator of metabolic activity in BC and suggest that reversal of the metabolic switch is a key mechanism of klotho-mediated tumor suppressor activities. Citation Format: Tami Rubinek, Ido Wolf. The tumor suppressor klotho: A master regulator of metabolism in breast cancer [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 5417. doi:10.1158/1538-7445.AM2017-5417

Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK's effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

High expression of partitioning defective 3-like protein is associated with malignancy in colorectal cancer.

Partitioning defective 3-like protein is a novel cell polarity protein. Recently, partitioning defective 3-like protein has been demonstrated with tumor-promoting function by disrupting tight junction, inhibiting tumor suppressor liver kinase B1, and maintaining mammary stem cells. For the first time, we studied partitioning defective 3-like protein expression in malignant colorectal cancer. We used immunohistochemistry scoring system to evaluate partitioning defective 3-like protein expression in 196 colorectal cancer tissues and 33 adjacent normal tissues. We found that colorectal cancer tissues had much stronger partitioning defective 3-like protein immunoreactivity than normal tissues, and colorectal cancer patients with positive partitioning defective 3-like protein expression were characterized with higher cancer stages, metastasis, poor tumor differentiation, larger tumor size, as well as high levels of colorectal cancer markers carcinoembryonic antigen and cancer antigen 19-9. Besides, partitioning defective 3-like protein overexpression was independently predictive of lower survival rate in colorectal cancer patients, even after adjusting the influence of cofactors. Moreover, we also found that partitioning defective 3-like protein was associated with rapid growing colorectal cancer, while knockdown of partitioning defective 3-like protein expression largely inhibited cancer cell proliferation. Our study provided the first evidence that partitioning defective 3-like protein was overexpressed in colorectal cancer and associated with disease malignancy. Also, partitioning defective 3-like protein may serve as a promising prognostic marker and a potential therapeutic target for colorectal cancer treatment. Further study is necessary to understand the regulatory mechanism of partitioning defective 3-like protein in colorectal cancer and the feasibility of its application in clinic.

LKB1 expression reverses the tumorigenicity of L02 cells.

The tumor-suppressor liver kinase B1 (LKB1), a highly conserved and ubiquitously expressed protein kinase, plays a critical role in tumorigenesis. In the present study, we revealed that human hepatic L02 cells had severely impaired endogenous LKB1 expression as gauged by western blot, northern blot and RT-PCR analyses. Stable ectopic expression of LKB1 in L02 cells resulted in decreased cell growth, hypophosphorylation of Rb, and marked attenuation of colony formation on soft agar. Inoculation of L02 cells into immunocompromised mice resulted in the development of subcutaneous tumors, which could be completely abrogated by ectopic LKB1 expression. The tumors that formed in the mouse model recapitulated the histopathological features of hepatocellular carcinoma under the microscope. Our results jointly suggest that severely compromised endogenous LKB1 expression in the L02 cell line may confer to L02 cells tumor-initiating capacities invivo and invitro, and ectopic LKB1 expression antagonizes the tumorigenic properties of L02 cells. Our findings imply that caution may be needed to interpret the results obtained on the widely used human hepatic L02 cell line. The L02 cell line may be a new model to define the cellular mechanisms of liver transformation, and to unravel the molecular mechanisms underlying the growth suppressive effect of LKB1.

Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

Abstract 1184: The tumor suppressor klotho: a master regulator of metabolism in breast cancer

Abstract One of the hallmarks of cancer is reprogramming of energy metabolism, characterized by a shift to aerobic glycolysis (Warburg effect). This metabolic switch provides cancer cells appropriate conditions required for enhanced proliferation and survival. A major regulator of this phenomenon is the IGF-1/Pi3K/AKT pathway. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We identified klotho as a tumor suppressor in breast and pancreatic cancers and recent data indicate it as a potent tumor suppressor in a wide array of malignancies. As klotho is a potent inhibitor of the IGF-1/Pi3K/AKT pathway in breast cancer, we hypothesized it may revert the metabolic switch in cancer cells. A master regulator of energy metabolism is the energy sensor enzyme, AMP-activated kinase (AMPK). We first analyzed the effect of klotho on AMPK and its down-stream effector acetyl CoA carboxylase (ACC) in MCF-7, T47D and MDA-MB-231 breast cancer cell lines. These cell lines represent different disease phenotypes. Overexpression of klotho, or treatment with the soluble protein, AMPK and ACC phosphorylation in all three cell lines. AMPK is activated by the tumor suppressor liver kinase B1 (LKB1) and elevated AMP/ATP ratio or by calcium/calmodulin-dependent protein kinase kinase (CAMKK) 1β. LKB1 was expressed in all cell lines, although lower expression was noted in MD-MB-231 cells. Expression of a dominant negative LKB1 prevented activation of AMPK by klotho. Furthermore, it decreased the ability of klotho to inhibit cell growth and migration, implying that klotho tumor suppressor activity may be LKB1 dependent. We next analyzed the effects of klotho on critical components of glucose metabolism. Treatment with soluble klotho reduced expression of the glucose transporter GLUT1 and the key glycolytic enzymes hexokinase2 (HK2), phosphofructokinase1 (PFK1), pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase kinase 1(PDK1). Finally, we examined the effects of klotho on specific metabolites and also conducted an NMR-based metabolic profiling. In accordance with the alterations in signaling pathways and levels of glycolytic enzymes, klotho reduced glucose uptake and inhibited lactate, pyruvate and 3-hydroxy-butyrate production. Furthermore, klotho also inhibited ATP production. Taken together, our data indicate klotho, for the first time, as a regulator of metabolic activity in breast cancer and suggest that reversal of the metabolic switch is a key mechanism of klotho-mediated tumor suppressor activities. Citation Format: Riva Shmulevich, Tammi Rubinstein, Taly Scherf, Ido Wolf, Tami Rubinek. The tumor suppressor klotho: a master regulator of metabolism in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2015-1184

Underexpression of LKB1 tumor suppressor is associated with enhanced Wnt signaling and malignant characteristics of human intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a rare and highly aggressive malignancy. In this study, we identified the presence of gene deletion and missense mutation leading to inactivation or underexpression of liver kinase B1 (LKB1) tumor suppressor and excluded the involvement of LKB1 gene hypermethylation in ICC tissues. Immunohistochemical analysis showed that LKB1 was underexpressed in a portion of 326 ICC tissues compared to their adjacent normal tissues. By statistical analysis underexpression of LKB1 in ICC tissues significantly correlated with poor survival and malignant disease characteristics in ICC patients. Moreover, we showed that knockdown of LKB1 significantly enhanced growth, migration, and invasion of three LKB1-competent ICC cell lines. Global transcriptional profiling analysis identified multiple malignancy-promoting genes, such as HIF-1α, CD24, Talin1, Vinculin, Wnt5, and signaling pathways including Hedgehog, Wnt/β-catenin, and cell adhesion as novel targets of LKB1 underexpression in ICC cells. Furthermore, knockdown of LKB1 gene expression dramatically enhanced Wnt/β-catenin signaling in ICC cells, while an inverse correlation between LKB1 and nuclear β-catenin was observed in ICC tissues. Our findings suggest a novel mechanism for ICC carcinogenesis in which LKB1 underexpression enhances multiple signaling pathways including Wnt/β-catenin to promote disease progression.

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell-cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

Abstract 1571: The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling

Abstract The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field. Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer. Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2014-1571

LKB1 and AMPK differentially regulate pancreatic β-cell identity.

Fully differentiated pancreatic β cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess (“glucotoxicity”) is implicated in this process, we sought here to identify the potential roles in β-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly β-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0–12-fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up-regulated β-cell “disallowed” genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to 3.4-fold (E<0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P=1.3×10−33) and hypoxia-regulated (HIF1; P=2.5×10−16) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain β-cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining β-cell function in some forms of diabetes.—Kone, M., Pullen, T. J., Sun, G., Ibberson, M., Martinez-Sanchez, A., Sayers, S., Nguyen-Tu, M.-S., Kantor, C., Swisa, A., Dor, Y., Gorman, T., Ferrer, J., Thorens, B., Reimann, F., Gribble, F., McGinty, J. A., Chen, L., French, P. M., Birzele, F., Hildebrandt, T., Uphues, I., Rutter, G. A. LKB1 and AMPK differentially regulate pancreatic β-cell identity.

Lkb1‐ a master tumor suppressor (LB108)

The small intestine is remarkable in that 109 epithelial cells are produced every twenty minutes, but cancer rarely develops. This is possible due to the serine/threonine kinase tumor suppressor Liver Kinase B1 (LKB1). Mutations in LKB1 were first linked to the disease Peutz‐Jeghers syndrome in which polyps grow and have a 93% chance of becoming cancerous. Mutations in LKB1 have been linked to several forms of cancer including small intestine, breast, lung, liver, and pancreas. LKB1 forms an active heterotrimer with pseudokinase: STRAD, and scaffolding protein: MO25. LKB1’s primary function is to regulate energy homeostasis through the phosphorylation of the t‐loop structure on adenosine monophosphate protein kinase (AMPK), which mediates the production of ATP when cells enter a state of energy stress. AMPK detects and reacts to differences in the AMP:ATP ratio. AMPK has also been shown to impact several cancer‐related processes. For example, the AMPK‐mTOR signaling pathway is important for inducing cell death within cancer cells. Possible future research includes finding methods of inducing LKB1 activity, which would stimulate the AMPK‐mTOR pathway and diminishes cancer cell proliferation. The CAPS SMART Team (Students Modeling a Research Topic) created physical models of the tumor suppressor protein LKB1 and its target AMPK using 3D printing technology.Grant Funding Source: Supported by grants from the NIH‐SEPA and NIH‐CTSA.

Downregulation of LKB1 suppresses Stat3 activity to promote the proliferation of esophageal carcinoma cells

The tumor suppressor liver kinase B1 (LKB1) encodes a serine/threonine kinase. The defect in LKB1 is the primary cause of Peutz-Jeghers syndrome (PJS). Inactivation of LKB1 by mutations or loss of LKB1 expression is associated with ovarian, lung and pancreatic cancer; however, the correlation between LKB1 and esophageal carcinoma remains unknown. Thus, quantitative PCR was performed to determine the clinical significance of LKB1 expression in 60 cases of esophageal cancer and its adjacent normal epithelium. LKB1 expression was observed to significantly downregulate the accompanying cancer progression, which was verified at the protein level by western blot analysis. Furthermore, the phosphorylated signal transducer and activator of transcription 3 (Stat3) level is reversibly associated with LKB1 expression. To determine the function of LKB1 in esophageal cancer, LKB1 expression is induced in TE1 esophageal cancer cells. The results show that LKB1 overexpression suppresses the proliferation of TE1 cells, downregulates the expression of cyclin D1 and Myc and represses Stat3 phosphorylation. Suppression of cell proliferation and cyclin D1 expression by LKB1 is fully inhibited by constitutively active Stat3C coexpression, suggesting that LKB1 inhibits esophageal cancer cell proliferation through suppression of Stat3 transaction. In conclusion, downregulation of LKB1 expression suppresses Stat3 activity that may promote tumor growth during esophageal cancer progression.

The LKB1 tumor suppressor differentially affects anchorage independent growth of HPV positive cervical cancer cell lines

Infection with high-risk human papillomaviruses is causally linked to cervical carcinogenesis. However, most lesions caused by high-risk HPV infections do not progress to cancer. Host cell mutations contribute to malignant progression but the molecular nature of such mutations is unknown. Based on a previous study that reported an association between liver kinase B1 (LKB1) tumor suppressor loss and poor outcome in cervical cancer, we sought to determine the molecular basis for this observation. LKB1-negative cervical and lung cancer cells were reconstituted with wild type or kinase defective LKB1 mutants and we examined the importance of LKB1 catalytic activity in known LKB1-regulated processes including inhibition of cell proliferation and elevated resistance to energy stress. Our studies revealed marked differences in the biological activities of two kinase defective LKB1 mutants in the various cell lines. Thus, our results suggest that LKB1 may be a cell-type specific tumor suppressor.

Dysregulation of mTOR activity through LKB1 inactivation

Mammalian target of rapamycin (mTOR) is aberrantly activated in many cancer types, and two rapamycin derivatives are currently approved by the Food and Drug Administration (FDA) of the United States for treating renal cell carcinoma. Mechanistically, mTOR is hyperactivated in human cancers either due to the genetic activation of its upstream activating signaling pathways or the genetic inactivation of its negative regulators. The tumor suppressor liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is involved in cell polarity, cell detachment and adhesion, tumor metastasis, and energetic stress response. A key role of LKB1 is to negatively regulate the activity of mTOR complex 1 (mTORC1). This review summarizes the molecular basis of this negative interaction and recent research progress in this area.

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44-436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.

Ly6/uPAR-Related Protein C4.4A as a Marker of Solid Growth Pattern and Poor Prognosis in Lung Adenocarcinoma

We have recently shown that the protein C4.4A is induced in early precursor lesions of pulmonary adenocarcinomas and squamous cell carcinomas. In the present study, we aimed at analyzing the impact of C4.4A on the survival of non-small cell lung cancer patients and determining whether its unexpected expression in adenocarcinomas could be attributed to a specific growth type (lepidic, acinar, papillary, micropapillary, solid). Sections from the center and periphery of the primary tumor, as well as N2-positive lymph node metastases, were stained by immunohistochemistry for C4.4A and scored semi-quantitatively for intensity and frequency of positive tumor cells. C4.4A score (intensity × frequency) in the tumor center was a highly significant prognostic factor in adenocarcinomas (n = 88), both in univariate (p = 0.004; hazard ratio [95% confidence interval] = 1.44 [1.12-1.85]) and multivariate statistical analysis (p = 0.0005; hazard ratio = 1.65 [1.24-2.19]), demonstrating decreasing survival with increasing score. In contrast, C4.4A did not provide prognostic information in squamous cell carcinomas (n = 104). Pathological stage was significant in both groups. In the adenocarcinomas, C4.4A expression was clearly associated with, but a stronger prognostic factor than, solid growth. The present results substantiate the potential value of C4.4A as a prognostic marker in pulmonary adenocarcinomas seen earlier in a smaller, independent patient cohort. Importantly, we also show that C4.4A is a surrogate marker for adenocarcinoma solid growth. Recent data suggest that C4.4A is negatively regulated by the tumor suppressor liver kinase B1, which is inactivated in some adenocarcinomas, providing a possible link to the impact of C4.4A on the survival of these patients.