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An extracellular matrix has been demonstrated to be one of the substances that promote tumor cells to become driven by external stimuli to change their biology or malignancy. The main component of extracellular matrix, chondroitin sulfate, has been confirmed as a factor in the progression of tumors. Nevertheless, a great deal remains unknown regarding the precise mechanisms that drive Chondroitin sulfate production and how these details are communicated to cancer cells. According to our previous studies, the main enzyme responsible for Chondroitin sulfate synthesis, carbohydrate sulfotransferase 11 (CHST11), plays a crucial role in lung cancer cell metastatic spread. Nevertheless, the relationship between CHST11 expression and regulatory mechanism in response to lung cancer treatment is unclear, especially for tyrosine kinase inhibitor (TKI). A clinical correlation analysis was used in this study to identify molecules which were highly correlated with the overexpression of CHST11 and identified GPI-anchored protein semaphorin 7A (SEMA7A) as a candidate. According to the TCGA database, expression of upregulated SEMA7A is correlated with poor prognosis and cellular malignancy in lung cancer patients. A SEMA7A-based gene correlation analysis finds that the affected gene ontology is highly similar to CHST11, particularly integrin signaling, which may contribute to Gefitinib resistance. A GSEA analysis reveals a significant effect of CHST11 overexpression on mitotic spindle processes, some of which molecules are clinically relevant to CHST11 or SEMA7A. These molecules have been reported to be involved in TKI resistance events such as MARCKS, MYO1E, and PDLIM5. Based on the TCGA database, these molecules and integrins also display significant clinical relevance in lung cancer patients. A CHST11-mediated increase in SEMA7A in the mitotic spindle process of cells may serve as one of the possible means of reducing TKI-induced mitotic cell death, and thereby promoting the ability of lung cancer cells to become resistant to TKIs. Hence, targeting the CHST11-SEMA7A axis may offer a therapeutic strategy option that could be used to target drug resistance in lung cancers. [Keywords: CHST11, SEMA7A, Integrin, Drug resistant, Lung cancer] (Reference: https://pubmed.ncbi.nlm.nih.gov/35985204/; https://pubmed.ncbi.nlm.nih.gov/36181245/) Citation Format: Chien-Hsiu Chris Li, Ming-Hsien Chan, Yu-Chan Chang, Michael Hsiao. CHST11 mediated SEMA7A expression contributes to TKI resistant in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3899.

The abundance and regulatory mechanisms of tumor microenvironment in gliomas remain unclear. We have previously demonstrated that aldolase c (ALDOC) has tumor suppressor function and prognostic value in the progression of glioma. In this study, we used immune infiltration algorithms to identify the immune response events that ALDOC modulates in gliomas and found that they were particularly strongly associated with cancer-associated fibroblasts (CAFs). Our transcriptomic and proteomic profiles infer that ALDOC is involved in the methylation status of N6-methyladenosine (m6A) and binds to KIAA1429, which in turn affects the assembly of the assembly of the m6A-METTL complex. We show that ALDOC can interact with KIAA1429 and trap it in the cytoplasm and then interfere with the modification of m6A. In malignant cells and ALDOC knockdown models, we observed loss of ALDOC function, KIAA1429 was responsible for the ‘writer’ of m6A and stabilized expression levels of various CAF-related features. Through the MeRIP-Seq/m6A-seq approach, we found that the RNA-binding protein, IGF2BP2, can be directly stabilized with multiple CAF-related genes. We examined ALDOC and KIAA1429, CAF-related genes (FAP, α-SMA, S100A4 and VIM) in available cohorts and tissue microarrays and were statistically inversely correlated. Furthermore, ALDOC colocalized with KIAA1429 and CAF genes in the surrounding microenvironment was significantly reduced in tissue sections from orthotopic animal models by multiplex immunohistochemistry. Keywords: Aldolase C, m6A modification, cancer-associated fibroblasts, gliomas Citation Format: Chien-Hsiu Li, Ming-Hsien Chan, Yu-Chan Chang. ALDOC interplays with KIAA1409 to reduce m6A modifications and cancer-associated fibroblast characteristics in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2352.

Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis, but is downregulated during lung cancer development and progression. The prognostic significance and functional consequences of FSTL1 downregulation in lung cancer are unclear. Here, reduced levels of FSTL1 were detected in various tumors compared with normal tissues and were associated with poor clinical outcome in patients with non–small cell lung cancer, particularly those with lung adenocarcinoma. FSTL1 expression negatively correlated with the metastatic potential of lung cancer cells. Antibody-based neutralization of extracellular FSTL1 increased cellular migration/invasion while addition of recombinant FSTL1 protein diminished the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, treatment with FSTL1 effectively prevented the metastatic progression of lung cancer cells in an orthotopic animal model. Mechanistically, FSTL1 directly bound to the proform of secreted phosphoprotein 1 (SPP1)/osteopontin, restraining proteolytic activation of SPP1, which led to inactivation of integrin/CD44-associated signaling and rearrangement of the actin cytoskeleton. Combined low expression of FSTL1 and high expression of SPP1 predicted a poorer prognosis for patients with lung cancer. This study highlights the novel interaction between FSTL1 and SPP1 and new opportunities to effectively target SPP1-driven metastatic cancers characterized by FSTL1 downregulation. Significance: These findings describe the novel interaction between FSTL1 and SPP1 and its role in the metastatic progression of lung adenocarcinoma.

Cancer metabolic reprogramming promotes tumorigenesis and metastasis; however, the underlying molecular mechanisms are still being uncovered. In this study, we show that the glycolytic enzyme aldolase A (ALDOA) is a key enzyme involved in lung cancer metabolic reprogramming and metastasis. Overexpression of ALDOA increased migration and invasion of lung cancer cell lines in vitro and formation of metastatic lung cancer foci in vivo. ALDOA promoted metastasis independent of its enzymatic activity. Immunoprecipitation and proteomic analyses revealed γ-actin binds to ALDOA; blocking this interaction using specific peptides decreased metastasis both in vitro and in vivo. Screening of clinically available drugs based on the crystal structure of ALDOA identified raltegravir, an antiretroviral agent that targets HIV integrase, as a pharmacologic inhibitor of ALDOA-γ-actin binding that produced antimetastatic and survival benefits in a xenograft model with no significant toxicity. In summary, ALDOA promotes lung cancer metastasis by interacting with γ-actin. Targeting this interaction provides a new therapeutic strategy to treat lung cancer metastasis. Significance: This study demonstrates the role of aldolase A and its interaction with γ-actin in the metastasis of non–small lung cancer and that blocking this interaction could be an effective cancer treatment.

Metabolism reprogramming and increased glycolysis of cancer are associated with rapid cell growth, motility, and resistance to drug treatment. Fructose-1, 6-bisphosphate (F-1,6-BP), an important product involvement in glucose metabolism, especially in cancer development. It is known that the essential enzymes of gluconeogenesis and glycolysis, fructose-1,6-bisphosphonates 1 (FBP1) and glycolytic enzyme aldolase A (ALDOA) expression are involved in the F-1,6-BP conversion process. Especially, increased expression of ALDOA has been identified to contribute to multiple human cancer development. However, the underlying molecular mechanism involvement of ALDOA and FBP1 on the switching of F-1,6-BP is still being uncovered. This study shows that the expression levels of ALDOA and FBP1 with an inversed correlation during the cancer progression, especially in lung adenocarcinoma and hepatocellular carcinoma. Immunoprecipitation and proteomics analyses revealed the candidates, including the anterior gradient 2 (AGR2), ubiquitin ligase gene cullin 4B (CUL4B), and telomeric repeat binding factor 1 (TERF1) are significantly correlated with ALDOA and FBP1 expression. We designed a specific peptide to interfere the interaction of candidates on both ALDOA and FBP1. Moreover, we evaluate the relative glucose metabolism events. Further, we raised the hypothesis that these candidates may serve as a modulator to regulate the conversion of F-1,6-BP and contribute to cancer progression, which can be used as a therapeutic target for further research. Citation Format: Chien Hsiu Li, Yu-Chan Chang, Michael Hsiao. The opposing regulation of ALDOA and FBP1 in Glucose metabolism contribute to cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2465.

Colorectal tumorigenesis during Epithelial-Mesenchymal transition (EMT) is associated with cancer metastasis and drug resistance. The small G-proteins family has been implicated in the regulation of cell cytoskeleton, cell movement, and vesicle transport. Small G-proteins family is divided into five main families. The Rab protein family is a member of the Ras superfamily of small G-proteins. We analyzed all probes of the Rab family members from microarray datasets and RNA-sequencing. We further calculated the EMT scores by using an algorithm. Our results showed that the RAB31 expression levels of the three probes are positively correlated with EMT score (Pearson rho=0.72, 0.71 and 0.68, respectively). Compared with other family members, RAB31 had most significantly value in colorectal cancer cell lines. We established several two-way model with cDNA and shRNAs of RAB31, respectively. Furthermore, we observed that the expression of epithelial type marker CDH1 was inhibited, and mesenchymal type markers SNAI1 and SNAI2 were upregulated in RAB31 overexpression models. Moreover, we found that the protein level of RAB31 was correlated with several clinicopathological parameters (N status, M status) and disease-free survival in tissue arrays through immunohistochemistry staining. According to The Cancer Genome Atlas Program (TCGA) and online meta-database, high RNA level of RAB31 was consistent in colorectal cancer (HR=1.38, n=466 and HR=1.29, n=482) and correlated with poor prognosis of patients. In addition, we identified that RAB31 may associated with IC50 of oxaliplatin and EGFR trafficking. We concluded that RAB31 regulates the sensitivity of oxaliplatin, an additional chemotherapy strategy and an independent prognostic factor for colorectal cancer. Citation Format: Chi-Long Chen, Yu-Chan Chang, Michael Hsiao. RAB31 triggers oxaliplatin resistance in colorectal cancer via epithelial-mesenchymal transition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1520.

Immunotherapy based on checkpoint inhibitors provides substantial clinical benefit, but only to a minority of cancer patients. Targeted immunological checkpoint pathways have demonstrated anti-tumor cytotoxicity in the treatment of refractory cancers. However, cancer cells release specific “drones”, such as small vesicles, which increase their ability in the blood and released the proteins such as PD-L1 to block the attack from T cells then they have a change to reach the tumor forming. To characterization of exosomes forming, trafficking and secretion can be a novel therapeutic approach to improve immunotherapy in cancer patients. The ATPase H+ Transporting V0 and V1 subunits (V-ATPase, also called ATP6 family) are critical for acidifying a variety of intracellular compartments and lysosomal exocytosis. Nevertheless, the dysfunctional roles of V-ATPases in glioblastoma has not yet been elucidated. For this purposed, we investigated V-ATPase members in lysosomal vesicles and found several candidates are key factors in regulating lysosomal exocytosis in glioblastoma cells. We established transcriptomics datasets by various glioblastoma cell lines, the results showed V-ATPases are highly expressed in malignant cell lines compared to benign or normal immortalized cells. Immunohistochemistry also performed a trend of consistency that increased of V-ATPase expression in tumor parts than normal adjacent tissues. The expression level of several V-ATPase subunits is also associated with clinical parameters and survival rate. Moreover, we observed the number of tumor infiltrating lymphocyte (TILs) was decreased in clinical cohorts and two-way cell models. Therefore, we established V-ATPases related proteomics datasets and found some interaction partners could stabilized protein structure of V-ATPase. Specific peptide can compete between V-ATPase and the interacting partners and further modulate TILs in glioblastoma. This approach can be a novel therapeutic strategy that combines immunotherapy to against glioblastoma in clinical patients. Citation Format: Yu-Chan Chang, Michael Hsiao. V-ATPase family regulates lysosomal exocytosis and neutralizes with tumor-infiltrating lymphocytes for glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1092.

Epigenetic correlates of the head and neck cancer may illuminate its pathogenic roots. Through a gene set enrichment analysis, we found that the oncogenic transcription factor RUNX2 is widely upregulated in the head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis, where it also predicts poor prognosis in patients with HNSCC. Enforced expression of ectopic RUNX2 promoted the metastatic capabilities of HNSCC, whereas RUNX2 silencing inhibited these features. Mechanistic investigations showed that manipulating levels of activin A (INHBA) could rescue or compromise the RUNX2-mediated metastatic capabilities of HNSCC cells. Furthermore, we found that miR-376c-3p encoded within the 3′-untranslated region of RUNX2 played a pivotal role in regulating RUNX2 expression in highly metastatic HNSCC cells, where it was downregulated commonly. Restoring miR-376c expression in this setting suppressed expression of RUNX2/INHBA axis along with metastatic capability. Clinically, we observed an inverse relationship between miR-376c-3p expression and the RUNX2/INHBA axis in HNSCC specimens. In summary, our results defined a novel pathway in which dysregulation of the RUNX2/INHBA axis due to miR-376c downregulation fosters lymph node metastasis in HNSCC. Cancer Res; 76(24); 7140–50. ©2016 AACR.

Lung cancer cells grow in-situ during low-oxygen situation and usually results in invasion and is frequently recurrent after therapy. However, the oxygen-mediated translation control of the metabolic events in lung cancer remains largely unknown. By using the integrated transcriptomics data and the shRNA library against glycolytic enzymes, here we identified that Aldolase A (ALDOA) up-regulation is highly correlated with migratory/invasive activity of lung cancer cells and poorer clinical outcomes. Significantly, knockdown of Aldolase A inhibited in vitro and in vivo migratory/invasive activity of lung cancer cell line with high ALDOA expression. Complementary, enforced overexpression of exogenous ALDOA promoted migration/invasion. Furthermore, microarray and Ingenuity pathway analyses reveal that ALDOA-driven lung cancer metastasis likely recruits Hypoxia-inducible factor 1 alpha (HIF1α)-associated signaling pathway. There were no significance changes at the mRNA level of HIF1α in ALDOA two-way models. However, HIF1α protein was found stabilized in hypoxic or normoxic condition through ALDOA overexpression to decrease prolyl hydroxylase (PHD) activity for hydroxylation-form of HIF1α to activate downstream genes including CXCR4, MMP9 in lung carcinogenesis. Interesting, HIF1α also was found to bind hypoxia-response element (HRE) domain of ALDOA to activate its transcription and form a positive feedback loop. IHC staining results further demonstrate that Aldolase A and HIF1α expression is positively correlated in clinical lung cancer tissues and the signature of high-level Aldolase A and HIF1α; expression strongly associates with worst survival probability in lung cancer patients. Our results not only unveil molecular mechanism by which Aldolase A promotes lung cancer metastasis via inducing the HIF1α activity but also provide a novel therapeutic strategy to combat malignant lung cancer via targeting ALDOA. Citation Format: Yu-Chan Chang, Michael Hsiao. Hypoxia-mediated translational control of hypoxia inducible factor 1 alpha through aldolase A feedback regulation in lung carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A22.

The dysregulated metabolism is an emerging hallmark of cancer. However, little is known about the metabolic requirements during cancer progression. Therefore, identifying metabolic genes that are critical for supporting cancer invasion/metastasis may provide novel targets for cancer diagnosis and therapy. By analyzing gene expression and proteomic profile of invasive lung cancer cells CL1-5 and its parental counterpart CL1-0, we identified Aldolase A (ALDOA), an enzyme of glycolysis pathway, is overexpressed in CL1-5 and is significantly associated with disease recurrence in lung cancer patient microarray datasets. Moreover, HIF-1α was identified as the most significant transcription factor in ALDOA-recurrence gene signature by IPA upstream regulator analysis. Furthermore, Immunohistochemistry analysis confirmed the expression of ALDOA was markedly higher in lung cancer tumors than in normal lung. High ALDOA expression was found significantly associated with lymph node involvement and disease recurrence. Moreover, overexpression of ALDOA was significantly associated with poor outcome in both overall and disease-free survival. Knockdown of ALDOA expression was found to suppress invasion potentials in highly invasive lung cancer cell lines while complementary overexpression of ALDOA in poorly invasive cells promoted it in vitro and in vivo. ALDOA overexpression also accelerated glucose consumption, ATP utilization and lactate production. Knockdown of ALDOA expression in CL1-5 abolished HIF-1α expression with concurrent Mesenchymal-to-Epithelial Transition (MET). Moreover, IHC analysis shows an inverse correlation between ALDOA and E-cadherin and combination of high ALDOA and low E-Cadherin showed an unfavorable outcome, in a sharp contrast, compared to patients with low ALDOA and high E-cadherin. Here, we identified ALDOA as a critical glycolytic enzyme involved in lung cancer progression and may be a marker for poor clinical outcome. Citation Format: Yu-Chan Chang. Aldolase A induces invasion/metastasis of lung cancer through modulating HIF1-α and is a marker for poor clinical outcome. [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 3365. doi:10.1158/1538-7445.AM2014-3365