AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Antiproliferative Activity of Piceamycin by Regulating Alpha-Actinin-4 in Gemcitabine-Resistant Pancreatic Cancer Cells.

Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G0/G1 phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.

Alpha-actinin 4 and tumorigenesis of hepatocellular carcinoma.

BACKGROUND: We examined the expression of alpha-actinin 4 (ACTN4) in hepatocellular carcinoma (HCC) tissues, to investigate the relationships between the expression of ACTN4 and the clinicopathological features of HCC patients. We have also analyzed the relevance of ACTN4 expression and survival time after HCC patients undergo surgery. METHODS: The expression of ACTN4 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry in 87 HCC patients. Statistical analyses were conducted with SPSS 22.0 software. RESULTS: qRT-PCR revealed that the expression of ACTN4 mRNA was significantly higher in HCC tissues than in adjacent tissues. Immunohistochemistry revealed that the positivity rate of ACTN4 was increased in HCC tissues compared with adjacent tissues (P=0.029). A Mann-Whitney U analysis revealed that positive ACNT4 expression was statistically significantly associated with the lymph node statuses of the HCC patients. Kaplan-Meier curves revealed that positive ACTN4 expression was inversely correlated with the 5-year survival times of HCC patients after surgery, and this relationship was statistically significant. CONCLUSIONS: ACTN4 plays an important role in the carcinogenesis and progression of HCC.

Actinin-4 protein overexpression as a predictive biomarker in adjuvant chemotherapy for resected lung adenocarcinoma.

AIM: Although several clinical trials demonstrated the benefits of platinum-combination adjuvant chemotherapy for stage II-IIIA lung adenocarcinoma, predictive biomarkers for the efficacy of such therapy have not yet been identified. We evaluated protein overexpression of actinin-4 as a predictive biomarker of the efficacy of adjuvant chemotherapy in resected lung adenocarcinoma. MATERIALS & METHODS: We measured actinin-4 protein levels in patients with completely resected stage II-IIIA lung adenocarcinoma using immunohistochemistry and then retrospectively compared survival between adjuvant chemotherapy and observation groups. RESULTS: A total of 148 eligible patients were classified into actinin-4 positive or negative cases by immunohistochemistry. In the former, patients with adjuvant chemotherapy survived significantly longer than those with observation (hazard ratio [HR]: 0.307; p = 0.028). But, no significant survival benefit was noted with adjuvant chemotherapy (HR: 0.926; p = 0.876) in the latter. CONCLUSION: This marker could predict the efficacy of adjuvant chemotherapy for resected lung adenocarcinoma patients.

The biological role of actinin-4 (ACTN4) in malignant phenotypes of cancer.

Invasion and metastasis are malignant phenotypes in cancer that lead to patient death. Cell motility is involved in these processes. In 1998, we identified overexpression of the actin-bundling protein actinin-4 in several types of cancer. Protein expression of actinin-4 is closely associated with the invasive phenotypes of cancers. Actinin-4 is predominantly expressed in the cellular protrusions that stimulate the invasive phenotype in cancer cells and is essential for formation of cellular protrusions such as filopodia and lamellipodia. ACTN4 (gene name encoding actinin-4 protein) is located on human chromosome 19q. ACTN4 amplification is frequently observed in patients with carcinomas of the pancreas, ovary, lung, and salivary gland, and patients with ACTN4 amplifications have worse outcomes than patients without amplification. In addition, nuclear distribution of actinin-4 is frequently observed in small cell lung, breast, and ovarian cancer. Actinin-4, when expressed in cancer cell nuclei, functions as a transcriptional co-activator. In this review, we summarize recent developments regarding the biological roles of actinin-4 in cancer invasion.

Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification.

BACKGROUND: Even if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. PATIENTS AND METHODS: The copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. RESULTS: Amplification (an increase in the copy number by ≥ 2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I-IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10(-5), Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. CONCLUSIONS: Amplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.