Myofibrillogenesis Regulator‐1 Regulates the Ubiquitin Lysosomal Pathway of Notch3 Intracellular Domain Through E3 Ubiquitin‐Protein Ligase Itchy Homolog in the Metastasis of Non‐Small Cell Lung Cancer

Abstract Myofibrillogenesis regulator‐1 (MR‐1) is a multifunctional protein involved in the development of various human tumors. The study is the first to report the promoting effect of MR‐1 on the development and metastasis of non‐small cell lung cancer (NSCLC). MR‐1 is upregulated in NSCLC and positively associated with poor prognosis. The overexpression of MR‐1 promotes the metastasis of NSCLC cells by stabilizing the expression of Notch3‐ICD (NICD3) in the cytoplasm through enrichment analysis, in vitro and in vivo experimental researches. And Notch3 signaling can upregulate many genes related to metastasis. The stabilizing effect of MR‐1 on NICD3 is achieved through the mono‐ubiquitin lysosomal pathway and the specific E3 ubiquitin ligase is Itchy homolog (ITCH). There is a certain interaction between MR‐1 and NICD3. Elevated MR‐1 can affect the level of ITCH phosphorylation, reduce its E3 enzyme activity, and thus lead to reduce the ubiquitination and degradation of NICD3. Interference with the interaction between MR‐1 and NICD3 can increase the degradation of NICD3 and impair the metastatic ability of NSCLC cells, which is a previously overlooked treatment option in NSCLC. In summary, interference with the interaction between MR‐1 and NICD3 in the progression of lung cancer may be a promising therapeutic target.


Myofibrillogenesis Regulator- 1
Figure S1 Construction of transgenic mice.(A) A schematic of the strategy for generating PNKD knock-in mice (PD-KI).(B) A schematic of the strategy for generating PNKD knockout mice (PD-KO).

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Figure S2 MR-1 promotes malignant progression of NSCLC in transgenic mice.(A) OS curve analysis for mice with the indicated genotypes at the indicated ages.Log-rank (Mantel-Cox) test.*p < 0.05.(B) Expression of epithelial mesenchymal transition (EMT) related proteins in lung tissue of transgenic mice with the indicated genotypes.(C) Immunohistochemical (IHC) of CK8 in lung tumor tissue and metastatic thyroid tissue of transgenic mice with the indicated genotypes.Scale bar, 400 μm.

Figure S3
Figure S3 The expression of MR-1 is positively correlated with lung cancer metastasis.(A) A GSEA analysis of gene expression profiles of NSCLC patients with high or low expression of PNKD in the TCGA lung cancer datasets.(B) An analysis of the correlation between PNKD and distant metastasis through TCGA lung cancer datasets.*p < 0.05.(C) Kaplan Meier analysis of the survival rate of 153 NSCLC patients with lymph node metastasis, who were divided into low or high subgroups based on median PNKD expression.*p < 0.05.(D, E) Representative images of wound healing experiments for the normal bronchial epithelial cell line (BEAS-2B) and seven NSCLC cell lines (H460, 95-D, H596, PC-9, H1975, A549, H1299).Scale bar, 500 μm.The data was shown as mean ± SEM (n = 3).(F) Spearman correlation analysis between the relative expression level of MR-1 and the 48 h scratch healing rate of eight cell lines.

Figure S7
Figure S7 Clustal X2.1 alignment of NICD sequences.Notch amino acid sequences were compiled from the NCBI GenBank database.After visualization by Genedoc, significant sequence alignment is shown for human NICD1-3.Lysine residues (K), which are potential targets for ubiquitin ligation, are highlighted to show their relative conservation among the three NICDs.At the right, amino acid residue counts reflect their positions throughout the protein sequence.

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Figure S8 (A) pCDNA3.1-HA-NICD3,pCMV3-WWP2 and pCMV3-DDK ubiquitin plasmids were co transfected into HEK-293T cells for 48 h, and the cell extracts were IP with anti-HA Ab.Ubiquitinated NICD3 was detected by Western blot.(B) After transient transfection of pCDNA3.1-MR-1,pCMV3-WWP2 plasmids in H1299 and H460 cells for 48 h, the effects of MR-1 and WWP2 on NICD3 expression were detected by Western blot.(C) HEK-293T cells were transfected with the indicated plasmids for 48 h.Cell extracts were IP with an anti-HA Ab.The interaction between WWP2 and NICD3 was detected by Western blot.

Figure S9
Figure S9 The binding of MR-1 and NICD3.(A) Endogenous binding of Notch3/NICD3 and MR-1 was detected in H1299 and H460 cells by Western blot.(B) Predictprotein platform predicted the transmembrane domain of MR-1.(C) Biology online software tool TMHMM Server, v.2.0 predicted the transmembrane domain of MR-1.(D) Schematic diagram of MR-1 region.(E) Detecting the availability of truncated plasmids by Western blot.(F) After transient transfection of pCMV6-DDK-MR-1 truncated plasmids and pCDNA3.1-HA-NICD3plasmid in HEK-293T cells for 48 h, the interaction of NICD3 and MR-1 region was detected by Western blot.(G) After transient transfection of pCMV6-DDK-MR-1 plasmids and pCMV6-ITCH plasmid in HEK-293T cells for 48 h, exogenous binding of NICD3 and MR-1 was detected in HEK-293T cells by Western blot.(H) After transient transfection of pCMV6-DDK-MR-1 truncated plasmids and pCMV6-ITCH plasmid in HEK-293T cells for 48 h, the interaction of NICD3 and MR-1 region was detected by Western blot.

Figure S13
Figure S13 Colony formation assays assessed evaluated the impact of TMP22 on the colony formation ability of H1299.Colony formation rate = (numbers of colonies / numbers of seeded cells) × 100%.The colony formation relative to control group was detected.The data was shown as mean ± SEM (n = 3).Unpaired t-test.**p < 0.01.

Figure S14
Figure S14A549 cells (1×10 7 cells) were injected intravenously into nude mice (n = 5 per group), followed by daily intravenous injection of normal saline five days later.The liver tissues after metastasis were shown.