Yi-Jing Hsiao

Opposite Effects of M1 and M2 Macrophage Subtypes on Lung Cancer Progression.

Macrophages in a tumor microenvironment have been characterized as M1- and M2-polarized subtypes. Here, we discovered the different macrophages’ impacts on lung cancer cell A549. The M2a/M2c subtypes promoted A549 invasion and xenograft tumor growth. The M1 subtype suppressed angiogenesis. M1 enhanced the sensitivity of A549 to cisplatin and decreased the tube formation activity and cell viability of A549 cells by inducing apoptosis and senescence. Different macrophage subtypes regulated genes involved in the immune response, cytoskeletal remodeling, coagulation, cell adhesion, and apoptosis pathways in A549 cells, which was a pattern that correlated with the altered behaviors of the A549 cells. Furthermore, we found that the identified M1/M2 gene signatures were significantly correlated with the extended overall survival of lung cancer patients. These results suggest that M1/M2 gene expression signature may be used as a prognostic indicator for lung cancer patients, and M1/M2 polarization may be a target of investigation of immune-modulating therapies for lung cancer in the future.

Tumor Suppressor HLJ1 Binds and Functionally Alters Nucleophosmin via Activating Enhancer Binding Protein 2α Complex Formation

HLJ1, a member of the heat shock protein 40 chaperone family, is a newly identified tumor suppressor that has been implicated in tumorigenesis and metastasis in non-small cell lung cancer. However, the mechanism of HLJ1 action is presently obscure. In this study, we report that HLJ1 specifically interacts with the nuclear protein nucleophosmin (NPM1), forming a multiprotein complex that alters the nucleolar distribution and oligomerization state of NPM1. Enforced accumulation of NPM1 oligomers by overexpression in weakly invasive but high HLJ1-expressing cells induced the activity of signal transducer and activator of transcription 3 (STAT3) and increased cellular migration, invasiveness, and colony formation. Furthermore, silencing HLJ1 accelerated NPM1 oligomerization, inhibited the activity of transcription corepressor activating enhancer binding protein 2alpha (AP-2alpha), and increased the activities of matrix metalloproteinase-2 (MMP-2) and STAT3. Our findings suggest that HLJ1 switches the role of NPM1, which can act as tumor suppressor or oncogene, by modulating the oligomerization of NPM1 via HLJ1-NPM1 heterodimer formation and recruiting AP-2alpha to the MMP-2 promoter.

JAG1 Is Associated with Poor Survival through Inducing Metastasis in Lung Cancer

JAG1 is a Notch ligand that plays a critical role in multiple signaling pathways. However, the functionality of JAG1 in non-small cell lung cancer (NSCLC) has not been investigated thoroughly. By comparison of gene transcripted RNA profiles in the cell line pair with differential invasion ability, we identified JAG1 as a potential metastasis enhancer in lung cancer. Ectopic expression of JAG1 on lung cancer cells enhanced cell migration and invasion as well as metastasis in vitro and in vivo. Conversely, knockdown of JAG1 with siRNA in highly invasive cancer cells led to the reduction of migration and invasion. In clinical analysis, JAG1 mRNA expression was higher in tumors than in adjacent normal tissues in 14 of 20 patients with squamous cell carcinoma (SCC). SCC patients with higher JAG1 transcription had poor overall survival than those with low-transcripted JAG1. Microarray analysis indicated that the enforced JAG1 transcription was associated with an elevated HSPA2 RNA transcription, which played a role in promoting cancer cell migration and invasion. In conclusion, this is the first study that demonstrated that JAG1 might act as a potential prognostic marker and JAG1/HSPA2 axis mediates lung cancer malignancy at least partly.

SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

HLJ1 is an endogenous Src inhibitor suppressing cancer progression through dual mechanisms

HLJ1 (DNAJB4), a DNAJ/Hsp40 chaperone, has emerged as a novel prognostic marker in lung cancers; however, the molecular contribution and functionality in neoplastic diseases remain to be established. This study demonstrated that HLJ1 inhibits epithelial–mesenchymal transition in vitro and reduces lung cancer metastasis in vivo. Using shRNA silencing and ectopic expression of HLJ1, we found that HLJ1 not only suppresses catalytic activity of Src but also downregulates the formation of oncogenic complexes associated with the EGFR, FAK and STAT3 signaling pathways. A screen of specimens from HLJ1-knockout mice and lung cancer patients validated that HLJ1 expression is inversely correlated with Src activity. Mechanistically, HLJ1 protein directly bound to catalytic and protein-binding domains of Src through its amino acid Y172 and the P301/P304 motif. Following Src-induced HLJ1 phosphorylation at Y172, HLJ1–Src interaction was elevated, resulting in Src inhibition and malignancy suppression. Interestingly, both Src-binding regions also occurred in other DNAJB family members and contributed to anti-invasive activities of DNAJB proteins. We conclude that HLJ1 is an endogenous Src inhibitor that can suppress cancer metastasis through complex interacting mechanisms. This HLJ1–Src complex might provide a promising molecular model for developing new anticancer strategies.

Abstract 1577: The Impact of M1/M2 Tumor Associated Macrophage (TAM) Polarization on Cancer Progression in NSCLC

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor microenvironment has important influences on tumor progression. One of the effectors is tumor-associated macrophages (TAMs) that are characterized as M1/M2 by their different immune responses, induced by Th1 and Th2. In our study, we polarized THP-1 into classically (M1) and alternatively (M2) activated macrophages and evaluated some different markers to distinguish M1 and M2 subtype (M2a, M2c). M1 macrophage expressed high IL-1, IL-6, IL-12, IL23, TNF-α, and a surface maker CCR7; M2a and M2c macrophages expressed high IL-10 and a surface maker CD206, but expressed CD23 and CD163, respectively. Microarray analysis was used to evaluate the alteration of gene expression of lung cancer cell line (A549) after coculture with different subtype TAMs (M0, M1, M2a and M2c). Bioinformatics analysis indicated that TAMs mainly alter cell cycle, coagulation and cell adhesion pathways in tumor cells. The M0, M2a and M2c promoted the invasion ability of A549 after coculture for 2days. Otherwise, M1 condition medium reduced cell viability and induced apoptosis of A549 but not H1299, the p53 null cell line. Moreover, the M2 macrophage condition medium but not M1 increased the drug resistance ability of A549 cells after long-term coculture. More importantly, in vivo tumorigenesis assay indicated that M2 can promote tumor growth and angiogenesis. Different subtypes of macrophages can modify behaviors of cancer cells and may contribute to tumor progression. Further studies in the detailed mechanisms involved in the interaction of TAMs and cancer cells will unveil the role of macrophages in tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2011-1577

Abstract 1564: Spanxa1 is a novel mediator of epithelial-mesenchymal transition in lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA SPANX (Sperm protein associated with the nucleus, X-linked) gene family express throughout spermatogenesis in agreement with a potential role in sperm development. It has been reported as a CTA (Cancer/Testis Antigen), which was highly expressed in testis and several cancers, such as gliobastoma, melanoma, testicular germ tumor and hematologic malignancies. Nevertheless, the function and molecular mechanism of SPANX involved in the cancer progression are greatly unknown. We found that the SPANX gene family member A1 (SPANXA1) was high-expressed in CL1-0, a less metastatic lung cancer cell line, and low-expressed in CL1-5, a more metastatic cell line. In vitro assays showed that the enforced expression of SPANXA1 suppressed the abilities of migration and invasion, and induced a dramatically mesenchymal - epithelial transition (EMT). In contrast, knock-down SPANXA1 could increase the cell mobility and invasiveness. To identify the underlying mechanism of SPANXA1, RNAs derived from SPANXA1-expressed cells and mock controls wrer assayed by gene expression microarrays and pathway analysis. The results showed that 1024 genes were differentially expressed in presence of SPANXA1 and six of top ten pathways belonged to EMT-related pathways analyzed by MetaCore software. The fact implied that SPANXA1 largely induces the EMT-related signalings. Quantitative RT-PCR assays were used to validate the altered expression of certain important genes induced by SPANXA1 in the identified pathways. The EMT regulating transcription factor, Slug, was decreased in SPAXA1-overexpressed cells and it also acts as a repressor for E-cadherin expression. We found that SPANXA1 mainly localized at nucleus and inhibited the formation of filopodia assayed by immunofluoresence staining. To explore the function of SPANXA1 in the nucleus, we performed Chromatin IP sequencing and the results elucidated that SPANXA1 might bind onto chromosome and regulate gene expressions. Furthermore, mouse model demonstrated that the distal metastasis was dramatically decreased in SPANXA1-overexpressed cells compared with the mock controls. These data suggest that SPANXA1 is a novel EMT modulator involved in lung cancer progression and implies the potential therapeutic role of SPANXA1 in the future. Citation Format: Shih-Chun Hsu, Yi-Jing Hsiao, Sing-Liang Yu, Ming-Shyue Lee. Spanxa1 is a novel mediator of epithelial-mesenchymal transition in lung cancer. [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 1564. doi:10.1158/1538-7445.AM2014-1564