SUMMARY Activation of inositol-requiring enzyme (IRE1α) is an indispensable step in remedying the cellular stress associated with lipid perturbation in the endoplasmic reticulum (ER) membrane. IRE1α is a single-spanning ER transmembrane protein possessing both kinase and endonuclease functions, and its activation can be fully achieved through the dimerization and/or oligomerization process. How IRE1α senses membrane lipid saturation remains largely unresolved. Using both computational and experimental tools, we systematically investigated the dimerization process of the transmembrane domain (TMD) of IRE1α and found that, with help of the serine 450 residue, the conserved tryptophan 457 residue buttresses the core dimerization interface of IRE1α-TMD. BiFC (bimolecular fluorescence complementation) experiments revealed that mutation on these residues abolished the saturated fatty acid-induced dimerization in the ER membrane and subsequently inactivated IRE1α activity in vivo. Therefore, our results suggest that the structural elements of IRE1α-TMD serve as a key sensor that detects membrane aberrancy.

Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here, we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1–XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1–XBP1 pathway. In addition, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. Implications: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.

<p>S1A. Plasmids containing single guide RNAs targeted against the IRE1 locus. B. XBP1 splicing assay to assay IRE1 endoribonuclease activity in Hep3B and MDA MB 231 cells. S2. CRISPR clones obtained for MDA-MB-231 cells. S3. CRISPR clones obtained for Hep3B cells. S4. CRISPR clones obtained for HepG2 cells. S5. Representative brightfield images of Boyden's chamber migration assay. S6. Representative brightfield images of Boyden's chamber migration assay. S7. Representative brightfield images of Boyden's chamber migration assay. S8. Heatmap of standardized DSP, ZEB1 or ZEB2 expression in the TCGA PANCAN dataset. S9. Scatter plots showing the correlation between DSP methylation and the expression of DSP, ZEB1 or ZEB2 in the TCGA. S10A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in MDA-MB-231 cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB2 at 80 nM for 24 hrs. S11. A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in Hep3B cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in Hep3B cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in Hep3B cells treated with siZEB2 at 80 nM for 24 hrs. S12. Positive ChIP control for ZEB1 binding in Hep3B, MDA-MB-231 and HepG2 cells in the CDH1 promoter. S13. Relative ZEB1 protein expression in PA or BSA treated (48 hr) WT- or IRE1-KO MDA-MB-231 cells. S14. Relative ZEB1 protein expression in PA or BSA treated (72 hrs) WT- or IRE1-KO Hep3B cells.</p>

<p>S1A. Plasmids containing single guide RNAs targeted against the IRE1 locus. B. XBP1 splicing assay to assay IRE1 endoribonuclease activity in Hep3B and MDA MB 231 cells. S2. CRISPR clones obtained for MDA-MB-231 cells. S3. CRISPR clones obtained for Hep3B cells. S4. CRISPR clones obtained for HepG2 cells. S5. Representative brightfield images of Boyden's chamber migration assay. S6. Representative brightfield images of Boyden's chamber migration assay. S7. Representative brightfield images of Boyden's chamber migration assay. S8. Heatmap of standardized DSP, ZEB1 or ZEB2 expression in the TCGA PANCAN dataset. S9. Scatter plots showing the correlation between DSP methylation and the expression of DSP, ZEB1 or ZEB2 in the TCGA. S10A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in MDA-MB-231 cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB2 at 80 nM for 24 hrs. S11. A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in Hep3B cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in Hep3B cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in Hep3B cells treated with siZEB2 at 80 nM for 24 hrs. S12. Positive ChIP control for ZEB1 binding in Hep3B, MDA-MB-231 and HepG2 cells in the CDH1 promoter. S13. Relative ZEB1 protein expression in PA or BSA treated (48 hr) WT- or IRE1-KO MDA-MB-231 cells. S14. Relative ZEB1 protein expression in PA or BSA treated (72 hrs) WT- or IRE1-KO Hep3B cells.</p>