Skin Cell Transformation Research Articles

UCP2 Upregulation Increases Fra-1 Expression and S-phase Cell Population without Decreasing Apoptosis during Skin Cell Transformation

Upregulation of uncoupling protein 2 (UCP2) is considered a prosurvival mechanism for cancer cells. This prosurvival function is thought to be mediated by UCP2’s uncoupling activity which reduces the production of superoxide in the mitochondria. However, exactly how highly expressed UCP2 regulates cell proliferation, cell cycle, and cell death during the early stage of tumorigenesis has not been studied thoroughly. For this purpose, we generated UCP2 stably overexpressed JB6 Cl-41 cells (a skin cell transformation model) and performed studies to answer the above questions. Our results demonstrated that UCP2 overexpression enhanced cell proliferation, activation of the oncoprotein Fra-1, anchorage-independent growth, 3D spheroids growth, and glucose uptake during skin cell transformation. Next, our results demonstrated that UCP2 overexpression resulted in marked decreases in the proportion of the cells in the G1 phase and an increase of cells in the S phase of the cell cycle, which was accompanied by increased expression of Cyclin E and Cdk2. Lastly, UCP2 overexpression did not enhance or suppress apoptosis during skin cell transformation, as indicated by Annexin V and active caspase 3/7 staining. Taken together, these data suggest that UCP2 upregulation mainly enhances the Fra-1 oncogenic pathway which drives cell proliferation, without inhibiting apoptosis during skin cell transformation.

DNA Methylome and Transcriptome Study of Triterpenoid CDDO in TPA-Mediated Skin Carcinogenesis Model.

Overexposure to ultraviolet radiation and environmental carcinogens drive skin cancer development through redox imbalance and gene mutation. Antioxidants such as triterpenoids have exhibited anti-oxidative and anti-inflammatory potentials to alleviate skin carcinogenesis. This study investigated the methylome and transcriptome altered by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or TPA with 2-cyano 2,3-dioxoolean-1,9-dien-28-oic acid (CDDO). The results show that CDDO blocks TPA-induced transformation dose dependently. Several differential expressed genes (DEGs) involved in skin cell transformation, while counteracted by CDDO, were revealed by differential expression analysis including Lyl1, Lad1, and Dennd2d. In CpG methylomic profiles, the differentially methylated regions (DMRs) in the promoter region altered by TPA while showing the opposite methylation status in the CDDO treatment group were identified. The correlation between DNA methylation and RNA expression has been established and DMRs showing inverse correlation were further studied as potential therapeutic targets. From the CpG methylome and transcriptome results, CDDO significantly restored gene expression of NAD(P)H:quinone oxidoreductase 1 (Nqo1) inhibited by TPA by decreasing their promoter CpG methylation. Ingenuity Pathways Analysis (IPA) shows that CDDO neutralized the effect of TPA through modulating cell cycles, cell migration, and inflammatory and immune response regulatory pathways. Notably, Tumor Necrosis Factor Receptor 2 (TNFR2) signaling was significantly downregulated by CDDO potentially contributing to prevention of TPA-induced cell transformation. Overall, incorporating the transcriptome, CpG methylome, and signaling pathway network, we reveal potential therapeutic targets and pathways by which CDDO could reverse TPA-induced carcinogenesis. The results could be useful for future human study and targets development for skin cancer.

UVB Drives Metabolic Rewiring and Epigenetic Reprograming and Protection by Sulforaphane in Human Skin Keratinocytes.

Sulforaphane (SFN) is a potent anticancer agent which could protect the skin from ultraviolet (UV) radiation-induced insults. Currently, the metabolic rewiring and epigenetic reprograming induced by UVB and the role of SFN in UVB-mediated skin cell transformation remain largely unknown. Herein, we study the metabolome, epigenome, and transcriptome of human keratinocytes (HaCaT cells) exposed to UVB with or without SFN using liquid chromatography-mass spectroscopy, DNA methylation sequencing, and RNA sequencing. UVB increases intracellular reactive oxygen species (ROS) and SFN enhances ROS acutely in post-UVB-exposed HaCaT cells. UVB and SFN alter multiple metabolites and metabolism-related signaling pathways. Pathway analysis shows that UVB impacts numerous signaling pathways including STAT3, inhibition of matrix metalloproteases, and TGF-β, among others. DNA/CpG methylation analysis shows that SFN could partially reverse some of the alterations of UVB-induced CpG methylome. Integrating RNA-seq and Methyl-seq data, starburst plots show the correlation of mRNA expression and CpG methylation status. The potential linkages between the metabolome, CpG methylome, and transcriptome suggest that metabolites produced during metabolism act as cofactors or substrates for catalytic epigenetic modification and transcriptional regulation. These results indicate that UVB drives metabolic rewiring, epigenetic reprograming, and phenotypic transcriptomic alterations and SFN would block or attenuate many of these aberrations, potentially contributing to the overall protective effect of SFN against UVB-induced skin damage.

Nfe2l2 Regulates Metabolic Rewiring and Epigenetic Reprogramming in Mediating Cancer Protective Effect by Fucoxanthin.

Fucoxanthin (FX) is a carotenoid with many pharmaceutical properties due to its antioxidant/anti-inflammatory and epigenetic effects. NFE2L2 is involved in the defense against oxidative stress/inflammation-mediated diseases, like anticancer effects elicited by phytochemicals including FX. However, the role of FX and NFE2L2 in metabolic rewiring, epigenomic reprogramming, and transcriptomic network in blocking pro-tumorigenic signaling and eliciting cancer-protective effects remains unknown. Herein, we utilized multi-omics approaches to evaluate the role of NFE2L2 and the impact of FX on tumor promoter TPA-induced skin cell transformation. FX blocked TPA-induced ROS and oxidized GSSG/reduced GSH in Nfe2l2wild-type(WT) but not Nfe2l2-knockdown (KD) cells. Both Nfe2l2 KD and TPA altered cellular metabolisms and metabolites which are tightly coupled to epigenetic machinery. The suppressive effects of FX on TPA-enhancedSAM/SAH was abrogated by Nfe2l2 KD indicating Nfe2l2 plays a critical role in FX-mediated metabolic rewiring and its potential consequences on epigenetic reprogramming. Epigenomic CpG methyl-seq revealed that FX attenuated TPA-induced differentially methylated regions (DMRs) of Uhrf1 and Dnmt1 genes. Transcriptomic RNA-seq showed that FX abrogated TPA-induced differentially expressed genes (DEGs) of Nfe2l2-related genes Nqo1, Ho1, and Keap1. Associative analysis of DEGs and DMRs identified that the mRNA expressions of Uhrf1 and Dnmt1 were correlated with the promoter CpG methylation status. Chromatin immunoprecipitation assay showed that FX restored Uhrf1 expression by regulating H3K27Me3 enrichment in the promoter region. In this context, FX/Nfe2l2's redox signaling drives metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the protection of TPA-induced JB6 cellular transformation skin cancer model. Graphical abstract.

UCP2 overexpression enhanced glycolysis via activation of PFKFB2 during skin cell transformation.

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane transporter which is often upregulated in human cancers. However, how this anion transporter affects tumorigenesis is not well understood. Using the skin cell transformation JB6 model, we demonstrated that UCP2 overexpression activated phosphofructokinase 2/fructose-2,6-bisphosphatase 2 (PFKFB2), a key regulator of glycolysis. In conjunction, upregulation of PFKFB2 expression correlated with elevated fructose 2,6-bisphosphate (Fru-2,6-P2) levels, 6-phosphofructo-1-kinase (PFK-1) activity, glucose uptake, and lactate production. Inhibiting PFKFB2 expression suppressed UCP2-mediated skin cell transformation, decreased cell proliferation, and enhanced mitochondrial respiration, while dampening aerobic glycolysis. The AKT signaling pathway was activated in the UCP2 overexpressed cells; furthermore, the activated AKT signaling contributed to the activation of PFKFB2. Whereas AKT inactivation blocked PFKFB2 activation, suggesting that AKT activation is an important step in PFKFB2 activation. Collectively, our data suggest that UCP2 is a critical regulator of cellular metabolism during cell transformation. Our data also demonstrate a potentially novel mechanism to understand UCP2's tumor-promoting role, which is through the AKT-dependent activation of PFKFB2 and thereby, the metabolic shift to glycolysis (the Warburg effect).

Abstract LB-153: Lotus leaf and Cassia seed extracts, through activating Nrf2 pathway, suppress TPA-induced mouse skin cell transformation

Abstract In traditional Chinese medicine, some Chinese herbs to be used as foods can protect and promote human health. Lotus (Nelumbo nucifera) leaves (LL) and Cassia (Cassia tora) seeds (CS) are often used as a food or an ingredient when cooking in Chinese region. LL and CS have been studied to exhibit some biological effects but there is no investigation to study their cancer chemopreventive potential. In this study, the extracts, including water extract (WE), water extraction residue ethanol extract (WREE) and ethanol extract (EE), of LL and CS were prepared. HepG2-C8 cell line, HepG2 cells stably transfected with Nrf2-ARE-luciferase plasmid, were used to screen out the extracts with cancer chemoprevetive potential. Our results showed that CS-WE, LL-WREE and LL-EE has better Nrf2-ARE-luciferase induction activity. Furthermore, we also found that these three extracts can inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent cell growth of JB6 P+ cells while up-regulating Nrf2 and Nrf2 downstream antioxidant/detoxification enzymes. These results suggested that Chinese herbal Lotus leaves and Cassia seeds to be used as foods might inhibit carcinogenesis through the regulation of Nrf2 pathway. Citation Format: Yen-Fan Chan, Zi-Han Lin, Pei-Chun Chen, Ping-Hua Sung, Zheng-Yuan Su. Lotus leaf and Cassia seed extracts, through activating Nrf2 pathway, suppress TPA-induced mouse skin cell transformation [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 LB-153. doi:10.1158/1538-7445.AM2017-LB-153

UCP2 upregulation promotes PLCγ-1 signaling during skin cell transformation.

Uncoupling protein 2 (UCP2), whose physiological role is to decrease mitochondrial membrane potential and reactive oxygen species (ROS) production, is often overexpressed in human cancers. UCP2 upregulation has recently been proposed as a novel survival mechanism for cancer cells. However, until now, how exactly UCP2 promotes tumorigenesis remains inconclusive. Based on a widely used skin cell transformation model, our data demonstrated that UCP2 differentially regulated ROS. UCP2 upregulation decreased superoxide whereas it increased hydrogen peroxide production with concomitant increase in the expression and activity of manganese superoxide dismutase (MnSOD), the primary mitochondrial antioxidant enzyme. Furthermore, hydrogen peroxide was responsible for induction of lipid peroxidation, and PLCγ-1 activation in UCP2 overexpressed cells. Additionally, PLCγ-1 activation enhanced skin cell transformation, and pharmacological, and siRNA mediated inhibition of PLCγ-1, markedly reduced colony formation, and 3D cell growth. Moreover, hydrogen peroxide scavenger, catalase, suppressed lipid peroxidation, and dampened PLCγ-1 activity. Taken together, our data suggest that (i) UCP2 is an important regulator of mitochondrial redox status and lipid signaling; (ii) hydrogen peroxide might mediate UCP2's tumor promoting activity; and (iii) pharmacological disruption of PLCγ-1 and/or hydrogen peroxide may have clinical utility for UCP2 overexpressed cancers.

Eupatilin inhibits EGF-induced JB6 cell transformation by targeting PI3K.

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that play fundamental roles in regulation of multiple signaling pathways, including cell proliferation, survival and cell cycle. Increasing evidence has shown that abnormal activation of PI3K pathway contributes to tumorigenesis and progression of various malignant tumors. Therefore, it is an attractive target of chemoprevention and chemotherapy. Eupatilin, a natural flavone compound extracted from Artemisia vulgaris, has antitumor and anti-inflammation efficacy. However, the direct target(s) of eupatilin in cancer chemoprevention are still elusive. In the present study, we reported eupatilin suppressed JB6 cell proliferation and its EGF-induced colony formation. Eupatilin attenuated phosphorylation of PI3K downstream signaling molecules. Downregulation ofcyclin D1 expression and arresting in G1 phase were induced through eupatilin treatment. Furthermore, we found it could bind to the p110α, a catalytic subunit of PI3K, by computational docking methods. Pull down assay outcomes also verified the binding of eupatilin with PI3K. Taken together, our results suggest that epatilin is a potential chemopreventive agent in inhibition of skin cell transformation by targeting PI3K.

Withaferin A suppresses the up-regulation of acetyl-coA carboxylase 1 and skin tumor formation in a skin carcinogenesis mouse model.

Withaferin A (WA), a natural product derived from Withania somnifera, has been used in traditional oriental medicines to treat neurological disorders. Recent studies have demonstrated that this compound may have a potential for cancer treatment and a clinical trial has been launched to test WA in treating melanoma. Herein, WA's chemopreventive potential was tested in a chemically-induced skin carcinogenesis mouse model. Pathological examinations revealed that WA significantly suppressed skin tumor formation. Morphological observations of the skin tissues suggest that WA suppressed cell proliferation rather than inducing apoptosis during skin carcinogenesis. Antibody Micro array analysis demonstrated that WA blocked carcinogen-induced up-regulation of acetyl-CoA carboxylase 1 (ACC1), which was further confirmed in a skin cell transformation model. Overexpression of ACC1 promoted whereas knockdown of ACC1 suppressed anchorage-independent growth and oncogene activation of transformable skin cells. Further studies demonstrated that WA inhibited tumor promotor-induced ACC1 gene transcription by suppressing the activation of activator protein 1. In melanoma cells, WA was also able to suppress the expression levels of ACC1. Finally, results using human skin cancer tissues confirmed the up-regulation of ACC1 in tumors than adjacent normal tissues. In summary, our results suggest that withaferin A may have a potential in chemoprevention and ACC1 may serve as a critical target of WA. © 2015 Wiley Periodicals, Inc.

Abstract 3053: Knockout of UCP2 suppresses skin carcinogenesis

Abstract Mitochondria are important and necessary components of cell organelles. In recent times, mitochondrial metabolism has regained attention due to the growing evidence suggesting that dysfunction of mitochondria could play a major role in cancer. Cancer cells that are actively proliferating have great demand for ATP. This huge demand for ATP is met by glycolysis, which is called the ‘Warburg Effect’. Although the Warburg effect has lately been challenged, dysfunction of the mitochondrial dependent processes is a characteristic feature of almost all tumor cells, which lead to higher oxidative stress, inhibition of apoptosis and enhanced cell proliferation; which are the hallmarks of tumor cells. Mitochondrial uncoupling proteins have gained enormous interest in the field of cancer metabolism due to their impact on mitochondrial dependent processes. Uncoupling proteins are a family of mitochondrial proteins present in the inner mitochondrial membrane whose physiological role is to decrease membrane potential and hence ATP production. One of the most interesting functions of UCPs is to decrease the generation of ROS. However, because of altered mitochondrial dependent processes, cancer cells have elevated levels of ROS production leading to a phenomenon called ‘oxidative stress’. To fight against this oxidative stress, UCP2 (a homologue of UCP family) has shown to be up-regulated in a number of human cancers. However, there is no direct evidence supporting UCP2 contributes to carcinogenesis. Considering this ability of UCP2 to modulate ROS, membrane potential and mitochondrial bioenergetics, an interesting question to us, was whether UCP2 is involved in the progression of cancer. Using UCP2 homozygous knockout mice and UCP2 overexpressed cells; our data have demonstrated that knockout of UCP2 suppressed skin carcinogenesis. Overexpression of UCP2 enhanced skin cell transformation and oncogenic activation. In addition, genipin, a known UCP2 inhibitor, was able to inhibit oncogenic activation during skin cell transformation, even in UCP2 overexpressed cells. Finally, although knockout of UCP2 increased the basal levels of protein oxidation, it did not enhance protein oxidation in mouse skin epidermal tissues during skin carcinogenesis. In summary, our data have provided direct evidence that UCP2 promotes carcinogenesis, and targeting UCP2 may serve as a translational approach for chemoprevention. Citation Format: Annapoorna Sreedhar, Chunjing Zhang, Yunfeng Zhao. Knockout of UCP2 suppresses skin carcinogenesis. [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 3053. doi:10.1158/1538-7445.AM2015-3053

Abstract 1256: The chemopreventive effects of carvedilol on skin carcinogenesis

Abstract Skin cancer, the majority of which is non-melanoma skin cancer (NMSC), is the most common type of cancer in the US. The rate of NMSC is increasing mainly due to depletion of the ozone layer and increased exposure to ultraviolet radiation. To reduce the risk of skin cancer, it is recommended to all individuals to limit sun exposure and use sunscreens. Despite these efforts, the incidence of NMSC continues to rise. Novel chemopreventive targets and nontoxic agents are needed. Epidemiological studies identified an association of psychosocial factors such as chronic stress or depression with cancer onset and progression. These factors are partly mediated by activation of the sympathetic nervous system which results in release of norepinephrine and epinephrine, the effects of which are mediated through the alpha- and beta-adrenergic receptors (alpha- and beta-AR). Further, the use of beta-AR antagonists (beta-blockers) has been associated with reduced cancer incidence. However, whether beta-blockers have chemopreventive activity or whether beta-ARs contribute to carcinogenesis is unknown. To determine whether agonizing or antagonizing beta-ARs affect skin cell transformation, we tested the beta-AR agonist isoproterenol (Iso) and antagonist carvedilol on epidermal growth factor (EGF)-mediated transformation of JB6 P+ cells (a skin cell model to study tumor promotion). The cells were treated with EGF (10 ng/mL) plus 0.1 µM or 1.0 µM Iso, yet no effect was observed. However, treatment with carvedilol dose-dependently inhibited the formation of EGF-induced soft agar colonies. Such effect was not caused by growth inhibition, because cytotoxicity and inhibition of cell proliferation were not observed for tested concentrations. Next, the expression of beta1-, 2- and 3-AR was determined in JB6 cells using RT-PCR. The results showed that only beta2-AR was detectable. We also determined whether beta-ARs are expressed in other types of cancer. In tissues derived from a rat model of 7, 12-dimethylbenz[α]anthracene (DMBA)-induced mammary cancer, expression of beta1- and 2-AR was up-regulated in tumors in comparison with normal tissues. The human cancer cell lines A549 and MDA-MB-231 also express beta2-AR. To determine the in vivo chemopreventive activity of carvedilol, we used a skin hyperplasia model in SENCAR mice induced by topical treatment with 100 nM DMBA twice a week for four weeks. Carvedilol was tested topically (5 and 10 µM) or orally (5 and 20 mg/kg), beginning at two weeks before the first dose of DMBA, three times a week, for six weeks. DMBA alone increased the epidermal thickness from the average of 77.8 + 13.6 nm in normal skin to 294 + 49.7 nm in DMBA-treated skin. Both topical and oral carvedilol treatment inhibited DMBA-induced hyperplasia (P < 0.05). In conclusion, these results suggest that beta-blockers may have chemopreventive activity. This study also suggests that beta-ARs may serve as a novel target for cancer prevention. Citation Format: Andy Chang, Mandy Liu, Steven Yeung, Jijun Hao, Cyrus Parsa, Robert Orlando, Bradley Andresen, Ying Huang. The chemopreventive effects of carvedilol on skin carcinogenesis. [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 1256. doi:10.1158/1538-7445.AM2014-1256

Requirement and Epigenetics Reprogramming of Nrf2 in Suppression of Tumor Promoter TPA-Induced Mouse Skin Cell Transformation by Sulforaphane

Nrf2 is a transcription factor that plays critical roles in regulating the expression of cellular defensive antioxidants and detoxification enzymes. However, the role of Nrf2 and Nrf2's epigenetics reprogramming in skin tumor transformation is unknown. In this study, we investigated the inhibitory role and epigenetics of Nrf2 on tumor transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 (JB6 P+) cells and the anticancer effect of sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables. After five days of treatment, SFN significantly inhibited TPA-induced JB6 cellular transformation and SFN enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein levels of the Nrf2 target genes HO-1, NQO1 and UGT1A1. Knockdown of Nrf2 attenuated the induction of Nrf2, HO-1 and NQO1 by SFN, enhanced TPA-induced colony formation and dampened the inhibitory effect of SFN on TPA-induced JB6 transformation. Epigenetics investigation using bisulfite genomic sequencing showed that SFN decreased the methylation ratio of the first 15 CpGs of the Nrf2 gene promoter, which was corroborated by increased Nrf2 mRNA expression. Furthermore, SFN strongly reduced the protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). SFN also inhibited the total histone deacetylase (HDAC) activity and decreased the protein expression of HDAC1, HDAC2, HDAC3 and HDAC4. Collectively, these results suggest that the anti-cancer effect of SFN against TPA-induced neoplastic transformation of mouse skin could involve the epigenetic reprogramming of anti-cancer genes such as Nrf2, leading to the epigenetic reactivation of Nrf2 and the subsequent induction of downstream target genes involved in cellular protection.

Isocitrate dehydrogenase 1 is downregulated during early skin tumorigenesis which can be inhibited by overexpression of manganese superoxide dismutase

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P-cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA-induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity.

Redox Factor-1 Mediates Inflammatory Response during Tumor Promotion in Skin Epidermal JB6 Cells

Recently, redox factor-1 (Ref-1) has received considerable attention as an enzyme for stimulating tumor cell growth. We hypothesized that Ref-1 is upregulated during the early stage of tumorigenesis. Utilizing both tumor promotion sensitive P+ and promotion resistant P? mouse skin epidermal JB6 cells, we found that Ref-1 expression was only induced in tumor promotion sensitive P+ cells following TPA treatment. Consistent with that, Ref-1 knockdown suppressed skin cell transformation. Interestingly, Ref-1 knockdown suppressed nuclear translocation of NF-kappaB subunit p65, and inhibited production of proinflammatory cytokines. These results suggest Ref-1 may promote early tumorigenesis and serve as a target for chemoprevention.

Enhancing mitochondrial respiration suppresses tumor promoter TPA-induced PKM2 expression and cell transformation in skin epidermal JB6 cells.

Differentiated cells primarily metabolize glucose for energy via the tricarboxylic acid cycle and oxidative phosphorylation, but cancer cells thrive on a different mechanism to produce energy, characterized as the Warburg effect, which describes the increased dependence on aerobic glycolysis. The M2 isoform of pyruvate kinase (PKM2), which is responsible for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells and may contribute to the Warburg effect. However, whether PKM2 plays a contributing role during early cancer development is unclear. In our studies, we have made an attempt to elucidate the effects of varying mitochondrial respiration substrates on skin cell transformation and expression of PKM2. Tumorigenicity in murine skin epidermal JB6 P+ (promotable) cells was measured in a soft agar assay using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter. We observed a significant reduction in cell transformation upon pretreatment with the mitochondrial respiration substrate succinate or malate/pyruvate. We observed that increased expression and activity of PKM2 in TPA-treated JB6 P+ cells and pretreatment with succinate or malate/pyruvate suppressed the effects. In addition, TPA treatment also induced PKM2 whereas PKM1 expression was suppressed in mouse skin epidermal tissues in vivo. In comparison with JB6 P+ cells, the nonpromotable JB6 P- cells showed no increase in PKM2 expression or activity upon TPA treatment. Knockdown of PKM2 using a siRNA approach significantly reduced skin cell transformation. Thus, our results suggest that PKM2 activation could be an early event and play a contributing role in skin tumorigenesis.

Involvement of the Paxillin Pathway in JB6 Cl41 Cell Transformation

Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)-dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser(63) and Ser(73). Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability.