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FIGURE 6 from Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail

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posted on 2024-03-08, 14:20 authored by Mysore S. Veena, Jungmo J. Gahng, Mustafa Alani, Albert Y. Ko, Saroj K. Basak, Isabelle Y. Liu, Kimberly J. Hwang, Jenna R. Chatoff, Natarajan Venkatesan, Marco Morselli, Weihong Yan, Ibraheem Ali, Karolina Elżbieta Kaczor-Urbanowicz, Bhavani Shankara Gowda, Patrick Frost, Matteo Pellegrini, Neda A. Moatamed, Sharon P. Wilczynski, Pascale Bomont, Marilene B. Wang, Daniel Sanghoon Shin, Eri S. Srivatsan

Inhibition of mouse lung metastasis with re-expression of gigaxonin. A, Table shows xenograft tumors formation by ME180 cells and lung metastasis by GAN edited clones. ME180 did not form lung metastasis. Greater than 50% reduced metastasis was seen in GAN lentiviral transfected cells compared with untransfected and control lentiviral transfected cells. B, H&E staining shows absence of tumor cells in the lung of parental ME180 cells after tail vein injection indicating absence of lung metastasis. While high metastasis (tumor cells infiltrating >50% of the lung, cells, bracketed and circled areas) is seen with GAN edited and control lentiviral transfected cells, a 50% reduction in metastasis (black circles) compared with the control virus transfected cells is observed in GAN lentiviral transfected cells. C and D, Statistically significant expression (3+, >90%, P < 0.01) of e-cadherin is seen in the epithelium of normal lung tissue after ME180 tail vein injection. GAN edited cells and control lentiviral transfected cells show low level expression (1+, 40%) in the lung metastatic tumor cells. GAN lentiviral transfected cells have higher expression (3+, >80%) in tumor cells (P < 0.01). E and F, The pneumocytes of normal lung tissue of ME180 injected mice shows nuclear Snail expression serving as an internal positive control. Lung tumors of GAN edited, and control lentiviral cell injected mice show higher nuclear expression. However, there is statistically significant reduced nuclear expression (P < 0.01) in GAN lentiviral injected lung tumor cells. The non-tumor cells of this sample (indicated by arrows) have weak cytoplasmic expression. Thus, an inverse relationship in the expression of e-cadherin and Snail is seen in gigaxonin overexpressing cells versus the GAN edited cells indicating a reduction in metastasis related to higher gigaxonin expression. Thicker arrows point to non-tumor cells and the thinner arrow points to tumor cells.

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VA | VA Greater Los Angeles Healthcare System (GLA)

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ARTICLE ABSTRACT

Gigaxonin is an E3 ubiquitin ligase that plays a role in cytoskeletal stability. Its role in cancer is not yet clearly understood. Our previous studies of head and neck cancer had identified gigaxonin interacting with p16 for NFκB ubiquitination. To explore its role in cancer cell growth suppression, we analyzed normal and tumor DNA from cervical and head and neck cancers. There was a higher frequency of exon 8 SNP (c.1293 C>T, rs2608555) in the tumor (46% vs. 25% normal, P = 0.011) pointing to a relationship to cancer. Comparison of primary tumor with recurrence and metastasis did not reveal a statistical significance. Two cervical cancer cell lines, ME180 and HT3 harboring exon 8 SNP and showing T allele expression correlated with higher gigaxonin expression, reduced in vitro cell growth and enhanced cisplatin sensitivity in comparison with C allele expressing cancer cell lines. Loss of gigaxonin expression in ME180 cells through CRISPR-Cas9 or siRNA led to aggressive cancer cell growth including increased migration and Matrigel invasion. The in vitro cell growth phenotypes were reversed with re-expression of gigaxonin. Suppression of cell growth correlated with reduced Snail and increased e-cadherin expression. Mouse tail vein injection studies showed increased lung metastasis of cells with low gigaxonin expression and reduced metastasis with reexpression of gigaxonin. We have found an association between C allele expression and RNA instability and absence of multimeric protein formation. From our results, we conclude that gigaxonin expression is associated with suppression of epithelial–mesenchymal transition through inhibition of Snail. Our results suggest that GAN gene exon 8 SNP T allele expression correlates with higher gigaxonin expression and suppression of aggressive cancer cell growth. There is downregulation of Snail and upregulation of e-cadherin through NFκB ubiquitination. We hypothesize that exon 8 T allele and gigaxonin expression could serve as diagnostic markers of suppression of aggressive growth of head and neck cancer.

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