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Supplementary Figure 2: Both E-cadherin and membrane β-catenin are downregulated in CRC.

Supplementary Table 2

Supplementary Table 1

Supplementary Figure Legends

Supplementary Figure Legends

Supplementary Table 2: Correlation between clinicopathologic features in CRC patients and the expression of PDLIM1, E-cadherin, membrane β-catenin, respectively.

Supplementary Figure 1: Loss of PDLIM1 promotes colorectal cancer (CRC) cells migration in vitro.

Supplementary Figure S3-6

Breast cancer is the most common cancer among women worldwide, yet successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to observed antitumor effects. However, the molecular mechanisms involved remain poorly understood. Here, we report a role for ivermectin in breast cancer suppression by activating cytostatic autophagy both in vitro and in vivo. Mechanistically, ivermectin-induced autophagy in breast cancer cells is associated with decreased P21-activated kinase 1 (PAK1) expression via the ubiquitination-mediated degradation pathway. The inhibition of PAK1 decreases the phosphorylation level of Akt, resulting in the blockade of the Akt/mTOR signaling pathway. In breast cancer xenografts, the ivermectin-induced cytostatic autophagy leads to suppression of tumor growth. Together, our results provide a molecular basis for the use of ivermectin to inhibit the proliferation of breast cancer cells and indicate that ivermectin is a potential option for the treatment of breast cancer. Cancer Res; 76(15); 4457–69. ©2016 AACR.

Supplementary Table 1:Clinicopathologic characteristics of CRC patients.

Supplementary Figure 3: PDLIM1 binds E-cadherin/β-catenin complex and attenuates EGF-induced EMT.

Supplementary Figure S1-2

Supplementary Table 3: Univariate analysis of overall survival at 5-year follow-up.

This file contains supplementary description regarding the materials and methods we used in this study, including lentivirus construction and cell transfection, real-time PCR, reagents, tissue microarray and immunohistochemistry, immunoprecipitation, western blot analysis, immunofluorescence, wound healing, transwell migration, and invasion assays.

Metastasis is a major cause of death in patients with colorectal cancer, and increasing evidence supports the contribution of the epithelial–mesenchymal transition (EMT) to cancer progression. The dissociation of the E-cadherin/β-catenin adhesion complex represents a key step in EMT and promotes cancer invasion and metastasis, but the upstream signaling pathways regulating this interaction are poorly understood. Here, we show that PDLIM1, a member of the PDZ and LIM protein family, was downregulated in highly metastatic colorectal cancer cells and liver metastases from colorectal cancer patients. We found that loss of PDLIM1 promoted the expression of EMT markers and increased the invasive and migratory properties of multiple colorectal cancer cell lines. Furthermore, PDLIM1 knockdown increased colon-derived liver metastasis in an orthotopic colorectal cancer model and promoted distant metastatic colonization in an experimental lung metastasis model. Mechanistic investigations revealed that PDLIM1 interacted with and stabilized the E-cadherin/β-catenin complex, thereby inhibiting the transcriptional activity of β-catenin and preventing EMT. Accordingly, PDLIM1 overexpression attenuated EMT of colorectal cancer cells. Moreover, the downregulation of PDLIM1 in colorectal cancer samples correlated with reduced E-cadherin and membrane β-catenin levels, and was associated with shorter overall survival. In conclusion, our study demonstrates that PDLIM1 suppresses EMT and metastatic potential of colorectal cancer cells by stabilizing β-catenin at cell–cell junctions, and its loss in metastatic tissues may represent a potential prognostic marker of aggressive disease. Cancer Res; 76(5); 1122–34. ©2015 AACR.

Supplementary Materials and Methods

Supplementary Figure S7-9

Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic target. In this study, we sought to identify glycolytic regulators in HCC that could be inhibited to prevent tumor progression and could also be monitored in vivo, with the goal of providing a theragnostic alternative to existing therapies.An orthotopic HCC rat model was used. Tumors were stimulated into a high-proliferation state by use of off-target liver ablation and were compared with lower-proliferating controls. We measured in vivo metabolic alteration in tumors before and after stimulation, and between stimulated tumors and control tumors using hyperpolarizedGlycolytic lactate flux increased within HCC tumors 3 days after tumor stimulation, correlating positively with tumor proliferation as measured with Ki67. This was associated with a shift towards aerobic glycolysis and downregulation of the pentose phosphate pathway detected by mass spectrometry. MRI-measured lactate flux was most closely coupled with PFKFB3 expression and was suppressed with direct inhibition using PFK15.Inhibition of PFKFB3 prevents glycolytic-mediated HCC proliferation, trackable by in vivo h

Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that TXNRD3, a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male reproduction. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We reveal that mitochondria develop more defined cristae during capacitation in wild type sperm. Absence of TXNRD3 alters redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

Hepatocellular carcinoma (HCC) is the 4th leading cause of cancer-related death worldwide. Despite therapeutic advancements, a significant proportion of patients progress, highlighting the need to improve diagnostic approaches to monitor therapy. Radiofrequency ablation (RFA), an accepted locoregional therapy for early HCC, can stimulate more aggressive tumor biology within under-treated or distant tumors. Increased glycolytic activity is a hallmark feature of cancer, but variations in metabolism and its link to tumor aggression can impact the sensitivity of HCC to therapies affecting these pathways. Tumor metabolic phenotypes can be characterized by hyperpolarized 13C MRI (h13C MRI), which can also be used to assess treatment response. In this study, we used orthotopic rat HCC models with two different cell lines N1S1 and MCA-RH7777, and analyzed their mRNA expression of glycolysis-related genes and in vivo glycolytic activity with h13C MRI. We then partially-treated tumors with radiofrequency ablation (RFA) and used h13C MRI to measure in vivo tumor metabolism before and 3 days after RFA to compare them to histologic and phenotypical measures of disease progression. N1S1 tumors grew only as single lesions, while MCA-RH7777 rapidly produced intra-hepatic and distal metastases. Glycolysis-related genes including HK2, HK3, PFKFB3, and MCT4 were highly expressed in N1S1, but not in MCA-RH7777, while GPT1 was highly expressed in MCA-RH7777 but not in N1S1. In vivo h13C MRI demonstrated significantly increased 13C lactate flux in N1S1 tumor compared to the background normal liver, while 13C lactate flux in MCA-RH7777 rat model did not, consistent with mRNA expression patterns. Tumor 13C lactate flux was significantly increased and 13C alanine flux was significantly decreased following partial RFA treatment to N1S1 tumors when compared to untreated tumors, which was associated with an increase in tumor Ki67 expression. However, no change was seen on h13C MRI between the RFA and control arms in MCA-RH7777 tumors, even though growth and total tumor burden progressed significantly more following RFA. In contrast to N1S1, there was also no difference between 13C lactate flux and 13C alanine flux within MCA-RH7777 tumors and the neighboring normal hepatic parenchyma, suggesting that glycolytic activity in these tumors did not drive RFA-stimulated progression. Targeting of glycolytic pathways as a potential therapy for HCC may therefore be limited to certain subtypes, and h13C MRI may be used to select for sensitivity. Citation Format: Qianhui Dou, Aaron K. Grant, Cody Callahan, Muneeb Ahmed, Leo L. Tsai. Variations in glycolytic activity of HCC subtypes as monitored by in vivo h 13C MRI [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 2807.