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Supplementary figure 1. Characterization of the cisplatin-resistant HNSCC cell lines. Supplementary figure 2. Genetic or pharmacological inhibition of G9a promotes cisplatin-induced apoptosis in SAS-CR cells.Supplementary figure 3. Depletion of G9a increases the sensitivity to cisplatin in OECM-1 cells.Supplementary figure 4. Depletion of G9a increases the platinum-DNA adduct accumulation. Supplementary figure 5. Inhibition of G9a by shRNA or enzymatic inhibitor decreases intracellular GSH levels. Supplementary figure 6. Multi-drug resistance to chemotherapeutic agents in G9a-overexpressed SAS cells. Supplementary figure 7. Effect of G9a on the expression of GSH synthesis enzymes. Supplementary figure 8. The expression of GSH synthesis enzymes in mouse xenograft tumors. Supplementary figure 9. ATF4 upregulates GCLC in cisplatin-resistant cells. Supplementary figure 10. Upregulated GCLC is required for G9a to promote cisplatin resistance.

Suppl. Figure 4. Upregulation of miR-519d and reciprocal downregulation of EphA4 in melanospheres. A, Quantitative RT-PCR analyses of miR-519d expression in SK-Mel-28 and Sk-Mel-90 adherent and sphere-forming cells. B, Decreased EphA4 expression level in sphere-forming cells, compared to the adherent cells. **, P < 0.01.

Supplementary Figures 1-8. Supplementary Figure 1: Stably expressing Keap1 by CL1-5 cells is accompanied by decreasing migratory and invasive abilities. Supplementary Figure 2: Manipulation of Keap1 expression (overexpression or knockdown) had no significant effect on the cell growth rate. Supplementary Figure 3: Keap1 suppresses the mobility of cancer cells. Supplementary Figure 4: Functional confirmation of Keap1 and Nrf2 expressions in CL1-0 and CL1-5 cells. Supplementary Figure 5: Cell proliferation was not affected by Nrf2 or S100P expression. Supplementary Figure 6: Representative IHC images of Nrf2 in lung cancer tissues from surgically resected specimens. Supplementary Figure 7: Keap1 regulates intra-pulmonary metastasis in a CL1-0 xenograft model. Supplementary Figure 8: Keap1 mediates the degradation of RhoA in CL1-5 cells.

Purpose: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2–related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1–Nrf2 axis in the progression of non–small cell lung cancer (NSCLC).Experimental Design: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1–Nrf2 axis in lung cancer mobility and progression.Results: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival of NSCLC patients. Moreover, manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100P was downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells.Conclusions: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression. Clin Cancer Res; 21(20); 4719–32. ©2015 AACR.

Materials and Methods

Supplementary Tables 1-3. Supplementary Table 1: Clinicopathologic characteristics of patients with associated expression of Keap1 protein in NSCLC. Supplementary Table 2: Correlation between levels of Keap1 and Nrf2 expression in lung cancer. Supplementary Table 3: Clinicopathologic characteristics of lung tumors with high and low expression of Nrf2 protein.

SUPPLEMENTARY INFORMATION Supplementary material and methods, Table S1, and Figure S1-S7 - This file contains Supplementary material and methods; Table S1. The Cox univariate and multivariate analyses of endocan and clinicopathological factors in overall survival of patients in NSCLC patients.; Supplementary Figure S1. The clinical value of endocan in lung adenocarcinoma.; Supplementary Figure S2. Correlation of endocan and EGFR expression in lung cancer patients.; Supplementary Figure S3. Endocan enhances EGF�induced activation of EGFR in a concentration-dependent manner.; Supplementary Figure S4. The JAK/STAT3 and ERK/ELK signaling regulates endocan promoter activity.; Supplementary Figure S5. The interaction of endocan and EGF/EGFR complex.; Supplementary Figure S6. Endocan peptide treatment inhibits the PC9 tumor growth in vivo.; Supplementary Figure S7. Endocan peptide treatment suppresses the activation of EGFR harboring L858R/T790M/C797S triple mutations.

Increasing evidence suggests that there is a unique cell subpopulation in melanoma that can form nonadherent melanospheres in serum-free stem cell medium, mimicking aggressive malignancy. Using melanospheres as a model to investigate progression mechanisms, we found that miR-519d overexpression was sufficient to promote cell proliferation, migration, invasion, and adhesion in vitro and lung metastatic capability in vivo. The cell adhesion receptor EphA4 was determined to be a direct target of miR-519d. Forced expression of EphA4 reversed the effects of miR-519d overexpression, whereas silencing of EphA4 phenocopied the effect of miR-519d. Malignant progression phenotypes were also affected at the level of epithelial-to-mesenchymal transition and the ERK1/2 signaling pathway inversely affected by miR-519d or EphA4 expression. In clinical specimens of metastatic melanoma, we observed significant upregulation of miR-519d and downregulation of EphA4, in the latter case correlated inversely with overall survival. Taken together, our results suggest a significant functional role for miR-519d in determining EphA4 expression and melanoma progression.Significance: These results suggest a significant role for miR-519d in determining expression of a pivotal cell adhesion molecule that may impact risks of malignant progression in many cancers. Cancer Res; 78(1); 216–29. ©2017 AACR.

Supplementary Figure legends (Figures 1-8)

Ovarian cancer spheroids constitute a metastatic niche for transcoelomic spread that also engenders drug resistance. Spheroid-forming cells express active STAT3 signaling and display stem cell–like properties that may contribute to ovarian tumor progression. In this study, we show that STAT3 is hyperactivated in ovarian cancer spheroids and that STAT3 disruption in this setting is sufficient to relieve chemoresistance. In an NSG murine model of human ovarian cancer, STAT3 signaling regulated spheroid formation and self-renewal properties, whereas STAT3 attenuation reduced tumorigenicity. Mechanistic investigations revealed that Wnt signaling was required for STAT3-mediated spheroid formation. Notably, the Wnt antagonist DKK1 was the most strikingly upregulated gene in response to STAT3 attenuation in ovarian cancer cells. STAT3 signaling maintained stemness and interconnected Wnt/β-catenin signaling via the miR-92a/DKK1–regulatory pathways. Targeting STAT3 in combination with paclitaxel synergistically reduced peritoneal seeding and prolonged survival in a murine model of intraperitoneal ovarian cancer. Overall, our findings define a STAT3–miR-92a–DKK1 pathway in the generation of cancer stem–like cells in ovarian tumors, with potential therapeutic applications in blocking their progression. Cancer Res; 77(8); 1955–67. ©2017 AACR.

Although new generations of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been developed for the treatment of patients with non–small cell lung cancer (NSCLC) with EGFR-mutant tumors, TKI resistance often returns as a result of additional EGFR mutations. In addition to seeking for next-generation EGFR-TKI, developing novel EGFR-targeting strategies may hold the key to overcome the vicious cycle of TKI resistance. Endocan is known as a receptor tyrosine kinase ligand enhancer in tumorigenesis, but the impact of endocan on EGFR-driven NSCLC progression remains unknown. In this study, higher endocan levels were found in lung tumors compared with cancer-free tissues and correlated with poor prognosis in patients with NSCLC harboring mutant EGFR; circulating endocan levels were also significantly higher in patients with mutant EGFR. Endocan facilitated EGFR signaling via direct binding and enhancing of the EGF–EGFR interaction and supported the growth of tumors driven by mutated EGFR. Activated EGFR in turn upregulated expression of endocan via JAK/STAT3 and ERK/ELK cascades, thus forming a positive regulatory loop of endocan-EGFR signaling. On the basis of the binding region between endocan and EGFR, we designed therapeutic peptides and demonstrated promising therapeutic effects in xenografts harboring EGFR mutations including TKI-resistant T790M. Together, our findings highlight the novel interaction between endocan and EGFR and new opportunities to effectively target endocan-EGFR regulatory axis in patients with TKI-resistant NSCLC.Significance:Endocan is a novel and critical regulator of EGF/EGFR signaling and serves as an alternative target of EGFR-TKI resistance in NSCLC.

Gene therapy trials in human breast, ovarian, and head and neck tumors indicate that adenovirus E1A can sensitize cancer cells to the cytotoxic effects of paclitaxel in vitro and in vivo. Resistance to paclitaxel has been reported to occur in cells expressing low levels of the Forkhead transcription factor FOXO3a. In this article, we report that FOXO3a is critical for E1A-mediated chemosensitization to paclitaxel. RNA interference–mediated knockdown of FOXO3a abolished E1A-induced sensitivity to paclitaxel. Mechanistic investigations indicated that E1A indirectly stabilized FOXO3a by acting at an intermediate step to inhibit a ubiquitin-dependent proteolysis pathway involving the E3 ligase βTrCP and the FOXO3a inhibitory kinase IKKβ. E1A derepressed this inhibitory pathway by stimulating expression of the protein phosphatase 2A (PP2A)/C protein phosphatases, which by binding to the TGF-β–activated kinase TAK1, inhibited its ability to activate IKKβ and, thereby, to suppress βTrCP-mediated degradation of FOXO3a. Thus, by stimulating PP2A/C expression, E1A triggers a signaling cascade that stabilizes FOXO3a and mediates chemosensitization. Our findings provide a leap forward in understanding paclitaxel chemosensitization by E1A, and offer a mechanistic rational to apply E1A gene therapy as an adjuvant for improving therapeutic outcomes in patients receiving paclitaxel treatment. Cancer Res; 71(21); 6878–87. ©2011 AACR.

Supplementary Methods, Figure Legends 1-12, Tables 1-5 from H3K9 Histone Methyltransferase G9a Promotes Lung Cancer Invasion and Metastasis by Silencing the Cell Adhesion Molecule Ep-CAM

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Supplementary Figures 1-12 from H3K9 Histone Methyltransferase G9a Promotes Lung Cancer Invasion and Metastasis by Silencing the Cell Adhesion Molecule Ep-CAM

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Although new generations of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been developed for the treatment of patients with non–small cell lung cancer (NSCLC) with EGFR-mutant tumors, TKI resistance often returns as a result of additional EGFR mutations. In addition to seeking for next-generation EGFR-TKI, developing novel EGFR-targeting strategies may hold the key to overcome the vicious cycle of TKI resistance. Endocan is known as a receptor tyrosine kinase ligand enhancer in tumorigenesis, but the impact of endocan on EGFR-driven NSCLC progression remains unknown. In this study, higher endocan levels were found in lung tumors compared with cancer-free tissues and correlated with poor prognosis in patients with NSCLC harboring mutant EGFR; circulating endocan levels were also significantly higher in patients with mutant EGFR. Endocan facilitated EGFR signaling via direct binding and enhancing of the EGF–EGFR interaction and supported the growth of tumors driven by mutated EGFR. Activated EGFR in turn upregulated expression of endocan via JAK/STAT3 and ERK/ELK cascades, thus forming a positive regulatory loop of endocan-EGFR signaling. On the basis of the binding region between endocan and EGFR, we designed therapeutic peptides and demonstrated promising therapeutic effects in xenografts harboring EGFR mutations including TKI-resistant T790M. Together, our findings highlight the novel interaction between endocan and EGFR and new opportunities to effectively target endocan-EGFR regulatory axis in patients with TKI-resistant NSCLC. Significance: Endocan is a novel and critical regulator of EGF/EGFR signaling and serves as an alternative target of EGFR-TKI resistance in NSCLC.

N-α-Acetyltransferase 10 protein (Naa10p) was reported to be an oncoprotein in androgen-dependent prostate cancer (PCa; ADPC) through binding and increasing transcriptional activity of the androgen receptor (AR). PCa usually progresses from an androgen-dependent to an androgen-independent stage, leading to an increase in the metastatic potential and an incurable malignancy. At present, the role of Naa10p in androgen-independent prostate cancer (AIPC) remains unclear. In this study, in silico and immunohistochemistry analyses showed that Naa10 transcripts or the Naa10p protein were more highly expressed in primary and metastatic PCa cancer tissues compared to adjacent normal tissues and non-metastatic cancer tissues, respectively. Knockdown and overexpression of Naa10p in AIPC cells (DU145 and PC-3M), respectively, led to decreased and increased cell clonogenic and invasive abilities in vitro as well as tumor growth and metastasis in AIPC xenografts. From the protease array screening, we identified a disintegrin and metalloprotease 9 (ADAM9) as a potential target of Naa10p, which was responsible for the Naa10p-induced invasion of AIPC cells. Naa10p can form a complex with ADAM9 to maintain ADAM9 protein stability and promote AIPC’s invasive ability which were independent of its acetyltransferase activity. In contrast to the Naa10p-ADAM9 axis, ADAM9 exerted positive feedback regulation on Naa10p to modulate progression of AIPC in vitro and in vivo. Taken together, for the first time, our results reveal a novel cross-talk between Naa10p and ADAM9 in regulating the progression of AIPC. Disruption of Naa10p–ADAM9 interactions may be a potential intervention for AIPC therapy.

Metastasis is the crucial mechanism to cause high mortality in lung cancer. Degradation of extracellular matrix (ECM) by proteolytic enzymes, especially matrix metalloproteinases (MMPs), is a key process for promoting cancer cell migration and invasion. Therefore, targeting MMPs might be a strategy for lung cancer metastasis suppression. Honokiol, a biological active component of Magnolia officinalis, has been indicated to suppress lung cancer tumorigenesis through epigenetic regulation. However, the regulation of MMPs‐mediated migration and invasion by honokiol through epigenetic regulation in lung cancer is still a mystery. In the present study, the migration and invasion ability of H1299 lung cancer was suppressed by noncytotoxic concentrations of honokiol treatment. The proteolytic activity of MMP‐9, rather than MMP‐2, was inhibited in honokiol‐treated H1299 cells. Honokiol‐inhibited MMP‐9 expression was through promoting MMP‐9 protein degradation rather than suppressing transcription mechanism. Furthermore, the expression of specific histone deacetylases 6 (HDAC6) substrate, acetyl‐α‐tubulin, was accumulated after honokiol incubation. The disassociation of MMP‐9 with hyper‐acetylated heat shock protein 90 (Hsp90) was observed resulting in MMP‐9 degradation after honokiol treatment. Meanwhile, honokiol‐suppressed MMP‐9 expression and invasion ability of H1299 lung cancer cells was rescued by HDAC6 overexpression. Accordingly, the results suggested that the suppression of migration and invasion activities by honokiol was through inhibiting HDAC6‐mediated Hsp90/MMP‐9 interaction and followed by MMP‐9 degradation in lung cancer., The suppression of migration and invasion activities of honokiol was through inhibiting HDAC6‐mediated Hsp90/MMP‐9 interaction and followed by MMP‐9 degradation in lung cancer.

An increasing number of enzymes involved in serine biosynthesis have been identified and correlated with malignant evolution in various types of cancer. Here we showed that the overexpression of phosphoserine aminotransferase 1 (PSAT1) is widely found in lung cancer tissues compared with nontumor tissues and predicts a poorer prognosis in patients with lung adenocarcinoma. PSAT1 expression was examined in a tissue microarray by immunohistochemistry. The data show that the knockdown of PSAT1 dramatically inhibits the in vitro and in vivo metastatic potential of highly metastatic lung cancer cells; conversely, the enforced expression of exogenous PSAT1 predominantly enhances the metastatic potential of lung cancer cells. Importantly, manipulating PSAT1 expression regulates the in vivo tumor metastatic abilities in lung cancer cells. Adjusting the glucose and glutamine concentrations did not alter the PSAT1-driven cell invasion properties, indicating that this process might not rely on the activation of its enzymatic function. RNA microarray analysis of transcriptional profiling from PSAT1 alternation in CL1-5 and CL1-0 cells demonstrated that interferon regulatory factor 1 (IRF1) acts as a crucial regulator of PSAT1-induced gene expression upon metastatic progression. Decreasing the IRF1-IFIH1 axis compromised the PSAT1-prompted transcriptional reprogramming in cancer cells. Our results identify PSAT1 as a key regulator by a novel PSAT1/IRF1 axis in lung cancer progression, which may serve as a potential biomarker and therapeutic target for the treatment of lung cancer patients.

Endocan is known to be a circulating dermatan sulfate proteoglycan that regulates endothelial cell function. Dysregulation of endocan expression is observed not only in the tumor vasculature but also in cancer cells. Accumulating evidence has revealed that disordered endocan facilitates cancer progression via enhancing cancer cell proliferation, cell mobility, and cancer stemness properties. Recently, various interacting proteins and diverse subcellular localizations of endocan were identified in cancer cells. Herein, we summarize the application of endocan in cancer diagnoses and prognoses using serum and tumor specimens. We further discuss that the aberrant molecular characteristics of endocan may be due to the mislocalization of endocan in cancer cells. Defining the specific cellular roles of endocan will provide a promising diagnostic factor and therapeutic target for cancer patients.

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers, which is the second most lethal tumor worldwide. Epigenetic deregulation is a common trait observed in HCC. Recently, increasing evidence suggested that the G9a histone methyltransferase might be a novel regulator of HCC development. However, several HCC cell lines were recently noted to have HeLa cell contamination or to have been derived from non-hepatocellular origin, suggesting that functional validation of G9a in proper HCC models is still required. Herein, we first confirmed that higher G9a messenger RNA and protein expression levels were correlated with poor overall survival (OS) and disease-free survival (DFS) rates of HCC patients from The Cancer Genome Atlas (TCGA) dataset and our recruited HCC cohort. In an in vitro functional evaluation of HCC cells, HCC36 (hepatitis B virus-positive (HBV+) and Mahlavu (HBV−)) cells showed that G9a participated in promoting cell proliferation, colony formation, and migration/invasion abilities. Moreover, orthotopic inoculation of G9a-depleted Mahlavu cells in NOD-SCID mice also resulted in a significantly decreased tumor burden compared to the control group. Furthermore, after surveying microRNA (miRNA, miR) prediction databases, we identified the liver-specific miR-122 as a G9a-targeting miRNA. In various HCC cell lines, we observed that miR-122 expression levels tended to be inversely correlated to G9a expression levels. In clinical HCC specimens, a significant inverse correlation of miR-122 and G9a mRNA expression levels was also observed. Functionally, the colony formation and invasive ability were attenuated in miR-122-overexpressing HCC cells. HCC patients with low miR-122 and high G9a expression levels had the worst OS and DFS rates compared to others. Together, our results confirmed the importance of altered G9a expression during HCC progression and discovered that a novel liver-specific miR-122-G9a regulatory axis exists.

Wnt/β‐catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/β‐catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell‐specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/β‐catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of β‐catenin to stabilize β‐catenin–TCF4 complex and facilitate the transactivation of Wnt/β‐catenin signaling targets. Accordingly, activated β‐catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/β‐catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.

Background/Aims: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy is a clinical option for non-small cell lung cancer (NSCLC) harboring activating EGFR mutations or for cancer with wild-type (WT) EGFR when chemotherapy has failed. MET receptor activation or MET gene amplification was reported to be a major mechanism of acquired resistance to EGFR-TKI therapy in NSCLC cells. Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that was shown to suppress metastasis of hepatocellular carcinoma via inhibiting MET activity. Until now, the biological function responsible for LECT2’s action in human NSCLC remains unclear. Methods: LECT2-knockout (KO) mice and NOD/SCID/IL2rgnull (NSG) mice were respectively used to investigate the effects of LECT2 on the tumorigenicity and metastasis of murine (Lewis lung carcinoma, LLC) and human (HCC827) lung cancer cells. The effect of LECT2 on in vitro cell proliferation was evaluated, using MTS and colony formation assays. The effect of LECT2 on cell motility was evaluated using transwell migration and invasion assays. An enzyme-linked immunosorbent assay was performed to detect secreted LECT2 in plasma and media. Co-immunoprecipitation and Western blot assays were used to investigate the underlying mechanisms of LECT2 in NSCLC cells. Results: Compared to WT mice, mice with LECT2 deletion exhibited enhanced growth and metastasis of LLC cells, and survival times decreased in LLC-implanted mice. Overexpression of LECT2 in orthotopic human HCC827 xenografts in NSG mice resulted in significant inhibition of tumor growth and metastasis. In vitro, overexpression of LECT2 or treatment with a recombinant LECT2 protein impaired the colony-forming ability and motility of NSCLC cells (HCC827 and PC9) harboring high levels of activated EGFR and MET. Mechanistic investigations found that LECT2 bound to MET and EGFR to antagonize their activation and further suppress their common downstream pathways: phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase. Conclusion: EGFR-MET signaling is critical for aggressive behaviors of NSCLC and is recognized as a therapeutic target for NSCLC especially for patients with acquired resistance to EGFR-TKI therapy. Our findings demonstrate, for the first time, that LECT2 functions as a suppressor of the progression of NSCLC by targeting EGFR-MET signaling.

Cancer cells encounter metabolic stresses such as hypoxia and nutrient limitations because they grow and divide more quickly than their normal counterparts. In response to glucose restriction, we found that nuclear translocation of the glycolic enzyme, pyruvate kinase M2 (PKM2), helped cancer cells survive under the metabolic stress. Restriction of glucose stimulated AMPK activation and resulted in co-translocation of AMPK and PKM2 through Ran-mediated nuclear transport. Nuclear PKM2 subsequently bound to Oct4 and promoted the expression of cancer stemness-related genes, which might enrich the cancer stem cell population under the metabolic stress. Nuclear PKM2 was also capable of promoting cancer metastasis in an orthotopic xenograft model. In summary, we found that cytosolic AMPK helped PKM2 carry out its nonmetabolic functions in the nucleus under glucose restriction and that nuclear PKM2 promoted cancer stemness and metastasis. These findings suggested a potential new targeting pathway for cancer therapy in the future.

KH-type splicing regulatory protein (KSRP) is a single-strand RNA binding protein which regulates mRNA stability either by binding to AU-rich elements (AREs) of mRNA 3'UTR or by facilitating miRNA biogenesis to target mRNA. Unlike its well-characterized function at the molecular level in maintaining RNA homeostasis, the role of KSRP in cancer progression remains largely unknown. Here we investigate the role of KSRP in non-small cell lung cancer (NSCLC). We first examined KSRP expression by immunohistochemistry in a cohort containing 196 NSCLC patients and observed a strong positive correlation between KSRP expression and survival of NSCLC patients. Multivariate analysis further identified KSRP as an independent prognostic factor. Manipulating KSRP expression significantly affected in vitro cell mobility and in vivo metastatic ability of NSCLC cells. Microarray analysis identified an ARE-containing gene, EGR3, as a downstream effector of KSRP in NSCLC. Interestingly, we found that KSRP decreased EGR3 mRNA stability in an ARE-independent manner. By screening KSRP-regulated miRNAs in NSCLC cells, we further found that miR-23a directly binds to EGR3 3'UTR, reducing EGR3 expression and thereby inhibiting NSCLC cell mobility. Our findings implicate a targetable KSRP/miR-23a/EGR3 signaling axis in advanced tumor phenotypes.

Transient chemotherapeutic response is a major obstacle to treating head and neck squamous cell carcinomas (HNSCC). Histone methyltransferase G9a has recently been shown to be abundantly expressed in HNSCC, and is required to maintain the malignant phenotype. In this study, we found that high G9a expression is significantly associated with poor chemotherapeutic response and disease-free survival in HNSCC patients. Similarly, G9a expression and enzymatic activity were elevated in cisplatin-resistant HNSCC cells. Genetic or pharmacologic inhibition of G9a sensitized the resistant cells to cisplatin, increasing cellular apoptosis. Mechanistic investigations indicated that G9a contributes to transcriptional activation of the glutamate-cysteine ligase catalytic subunit (GCLC), which results in upregulation of cellular glutathione (GSH) and drug resistance. In addition, we observed a significant positive correlation between G9a and GCLC expression in tumors of HNSCC patients. Taken together, our findings provide evidence that G9a protects HNSCC cells against chemotherapy by increasing the synthesis of GSH, and imply G9a as a promising target for overcoming cisplatin resistance in HNSCC. Mol Cancer Ther; 16(7); 1421–34. ©2017 AACR.

Carbonic anhydrase IX (CA9) expression level has been considered as a poor prognostic factor in hepatocellular carcinoma (HCC) patients. However, the judging criteria of CA9 level is hard to define for potential clinical applications. Unlike CA9 expression level, CA9 polymorphism is poorly documented in HCC. Here, we found that people carry A allele at CA9 rs1048638, a 3′UTR SNP, has higher risk of HCC. rs1048638-CA correlates with advanced stages, larger tumor sizes, more vascular invasion, and shorter survival of HCC patients. A allele at CA9 rs1048638 impairs miR-34a, a tumor suppressor miRNA in HCC, binding to CA9 3′UTR and desensitizes CA9 mRNA to miR-34a-dependent RNA degradation. CA9 expression levels were also correlated with miR-34a levels and rs1048638 genotypes in HCC patients. rs1048638 influences HCC risk and progression through effects on miR-34a-targeted CA9 expression in HCC. In conclusion, genetic variations of the CA9 3′UTR play important roles in regulating CA9 expression and cancer progression, which is a novel determinant and target for HCC metastasis and prognosis.

An increasing number of enzymes involved in serine biosynthesis have been identified and correlated with malignant evolution in various types of cancer. Here we showed that the overexpression of phosphoserine aminotransferase 1 (PSAT1) is widely found in lung cancer tissues compared with nontumor tissues and predicts a poorer prognosis in patients with lung adenocarcinoma. PSAT1 expression was examined in a tissue microarray by immunohistochemistry. The data show that the knockdown of PSAT1 dramatically inhibits the in vitro and in vivo metastatic potential of highly metastatic lung cancer cells; conversely, the enforced expression of exogenous PSAT1 predominantly enhances the metastatic potential of lung cancer cells. Importantly, manipulating PSAT1 expression regulates the in vivo tumor metastatic abilities in lung cancer cells. Adjusting the glucose and glutamine concentrations did not alter the PSAT1-driven cell invasion properties, indicating that this process might not rely on the activation of its enzymatic function. RNA microarray analysis of transcriptional profiling from PSAT1 alternation in CL1-5 and CL1-0 cells demonstrated that interferon regulatory factor 1 (IRF1) acts as a crucial regulator of PSAT1-induced gene expression upon metastatic progression. Decreasing the IRF1-IFIH1 axis compromised the PSAT1-prompted transcriptional reprogramming in cancer cells. Our results identify PSAT1 as a key regulator by a novel PSAT1/IRF1 axis in lung cancer progression, which may serve as a potential biomarker and therapeutic target for the treatment of lung cancer patients.

Our study elucidated the clinical and prognostic significance of LCN2 in OSCC among the Taiwanese population. Moreover, we observed that LCN2 suppresses cell motility by downregulating CAIX protein level via HIF-1 alpha-mediated transcriptional regulation and miR-4505-mediated post-transcriptional regulation.Lipocalin 2 (LCN2), a secreted glycoprotein, is up- or downregulated in different human cancers. At present, the functional role of LCN2 in the progression of oral squamous cell carcinoma (OSCC), which accounts for most head and neck cancers, remains poorly understood, particularly with respect to its involvement in invasion and metastasis. In this study, we observed that LCN2 expression decreased in patients with OSCC and lymph node metastasis compared with that in patients without metastasis. A higher LCN2 expression correlated with the survival of patients with OSCC. Furthermore, LCN2 overexpression in OSCC cells reduced in vitro migration and invasion and in vivo metastasis, whereas its silencing induced an increase in cell motility. Mechanistically, LCN2 inhibited the cell motility of OSCC cells through hypoxia-inducible factor (HIF)-1 alpha-dependent transcriptional inhibition of the carbonic anhydrase IX (CAIX). CAIX overexpression relieved the migration inhibition imposed by LCN2 overexpression in OSCC cells. Moreover, a microRNA (miR) analysis revealed that LCN2 can suppress CAIX expression and cell migration through miR-4505 induction. Examination of tumour tissues from patients with OSCC and OSCC-transplanted mice revealed an inverse correlation between LCN2 and CAIX expression. Furthermore, patients with LCN2(strong)/CAIX(weak) revealed the lowest frequency of lymph node metastasis and the longest survival. Our findings suggest that LCN2 suppresses tumour metastasis by targeting the transcriptional and post-transcriptional regulation of CAIX in OSCC cells. LCN2 overexpression may be a novel OSCC treatment strategy and a useful biomarker for predicting OSCC progression.

N-α-Acetyltransferase 10 protein (Naa10p) mediates N-terminal acetylation of nascent proteins. Oncogenic or tumor suppressive roles of Naa10p were reported in cancers. Here, we report an oncogenic role of Naa10p in promoting metastasis of osteosarcomas. HigherNAA10transcripts were observed in metastatic osteosarcoma tissues compared to non-metastatic tissues and were also correlated with a worse prognosis of patients. Knockdown and overexpression of Naa10p in osteosarcoma cells respectively led to decreased and increased cell migratory/invasive abilities. Re-expression of Naa10p, but not an enzymatically inactive mutant, relieved suppression of the invasive abilityin vitroand metastasisin vivoimposed by Naa10p-knockdown. According to protease array screening, we identified that matrix metalloproteinase (MMP)-2 was responsible for the Naa10p-induced invasive phenotype. Naa10p was directly associated with MMP-2 protein through its acetyltransferase domain and maintained MMP-2 protein stability via NatA complex activity. MMP-2 expression levels were also significantly correlated with Naa10p levels in osteosarcoma tissues. These results reveal a novel function of Naa10p in the regulation of cell invasiveness by preventing MMP-2 protein degradation that is crucial during osteosarcoma metastasis.

Secondary mutation of epidermal growth factor receptor (EGFR) resulting in drug resistance is one of the most critical issues in lung cancer therapy. Several drugs are being developed to overcome EGFR tyrosine kinase inhibitor (TKI) resistance. Here, we report that pyruvate kinase M2 (PKM2) stabilized mutant EGFR protein by direct interaction and sustained cell survival signaling in lung cancer cells. PKM2 silencing resulted in markedly reduced mutant EGFR expression in TKI-sensitive or -resistant human lung cancer cells, and in inhibition of tumor growth in their xenografts, concomitant with downregulation of EGFR-related signaling. Mechanistically, PKM2 directly interacted with mutant EGFR and heat-shock protein 90 (HSP90), and thus stabilized EGFR by maintaining its binding with HSP90 and co-chaperones. Stabilization of EGFR relied on dimeric PKM2, and the protein half-life of mutant EGFR decreased when PKM2 was forced into its tetramer form. Clinical levels of PKM2 positively correlated with mutant EGFR expression and with patient outcome. These results reveal a previously undescribed non-glycolysis function of PKM2 in the cytoplasm, which contribute to EGFR-dependent tumorigenesis and provide a novel strategy to overcome drug resistance to EGFR TKIs.

Purpose: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2–related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1–Nrf2 axis in the progression of non–small cell lung cancer (NSCLC). Experimental Design: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1–Nrf2 axis in lung cancer mobility and progression. Results: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival of NSCLC patients. Moreover, manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100P was downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells. Conclusions: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression. Clin Cancer Res; 21(20); 4719–32. ©2015 AACR.

G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42.

Honokiol, an active compound derived from Magnolia spp . bark, possesses chemopreventive properties in many cancer cell models. However, the chemopreventive mechanism of honokiol in lung cancer cells is still a mystery. We examined histone deacetylase 6 (HDAC6)-mediated epidermal growth factor receptor (EGFR) stability in honokiol-treated lung cancer cells. The results showed that honokiol induced G1 growth arrest was through down-regulation of EGFR expression and thereafter downstream signaling pathways. HDAC6 activity was directly inhibited via honokiol at IC 50 about 23.55 ± 1.18 µM. Inhibition of HDAC6 activity via honokiol was followed by disrupting HDAC6 and heat shock protein 90 (Hsp90) association and resulting in Hsp90 hyper-acetylation. Meanwhile, hyper-acetylated Hsp90 had been found to disassociate with EGFR and followed by EGFR degradation. Taken together, these results suggested that interruption of EGFR stability by honokiol was through inhibiting HDAC6 activity and consequently suppressing Hsp90 chaperon function in lung cancer cells.

No effective targeted therapy exists for esophageal squamous cell carcinoma (ESCC), the major cell type of esophageal cancer. The pleiotropic cytokine interleukin (IL)-6 is associated with adverse prognosis of some cancers, and the open reading frame of IL-6 contains an miR-608 microRNA-targeted site. We investigated the correlation of circulating IL-6 levels with prognosis and with the mir608:rs4919510 genetic polymorphism in ESCC. A total of 213 patients with primary ESCC were enrolled. Plasma IL-6 levels of ESCC patients were analyzed by enzyme-linked immunosorbent assay (ELISA). The patients’ genotypes of mir608:rs4919510 were analyzed using the MassARRAY system, and functional assays were performed by transient overexpression in cells. The cytotoxicity of IL-6 signaling blockers in ESCC cells was analyzed by MTT assay. We found that plasma IL-6 levels significantly correlated with overall survival (p = 0.019), disease recurrence (p = 0.003), and postoperative complications (p =0.002). Patients with the GG genotype of mir608:rs4919510 had a 4.56-fold increased risk of high expression of IL-6 compared with patients with the CC genotype (odds ratio 4.56, 95% confidence interval 1.87–11.09; p =0.001). Transient overexpression of the miR-608 C (miR-608_C) and G variants (miR-608_G) in cancer cells revealed that the miR-608_G variant was less efficient in regulating the expression of IL-6 compared with miR-608_C. Finally, the IL-6 signaling blocker ruxolitinib exhibited effective cytotoxicity in ESCC cells. The results of this study provide a novel direction for a biomarker-based targeted therapy for ESCC.