TMEM65 promotes gastric tumorigenesis by targeting YWHAZ to activate PI3K-Akt-mTOR pathway and is a therapeutic target

Abstract

Copy number alterations are crucial for the development of gastric cancer (GC). Here, we identified Transmembrane Protein 65 (TMEM65) amplification by genomic hybridization microarray to profile copy-number variations in GC. TMEM65 mRNA level was significantly up-regulated in GC compared to adjacent normal tissues, and was positively associated with TMEM65 amplification. High TMEM65 expression or DNA copy number predicts poor prognosis (P < 0.05) in GC. Furtherly, GC patients with TMEM65 amplification (n = 129) or overexpression (n = 78) significantly associated with shortened survival. Ectopic expression of TMEM65 significantly promoted cell proliferation, cell cycle progression and cell migration/invasion ability, but inhibited apoptosis (all P < 0.05). Conversely, silencing of TMEM65 in GC cells showed opposite abilities on cell function in vitro and suppressed tumor growth and lung metastasis in vivo (all P < 0.01). Moreover, TMEM65 depletion by VNP-encapsulated TMEM65-siRNA significantly suppressed tumor growth in subcutaneous xenograft model. Mechanistically, TMEM65 exerted oncogenic effects through activating PI3K-Akt-mTOR signaling pathway, as evidenced of increased expression of key regulators (p-Akt, p-GSK-3β, p-mTOR) by Western blot. YWHAZ (Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase) was identified as a direct downstream effector of TMEM65. Direct binding of TMEM65 with YWHAZ in the cytoplasm inhibited ubiquitin-mediated degradation of YWHAZ. Moreover, oncogenic effect of TMEM65 was partly dependent on YWHAZ. In conclusion, TMEM65 promotes gastric tumorigenesis by activating PI3K-Akt-mTOR signaling via cooperating with YWHAZ. TMEM65 overexpression may serve as an independent new biomarker and is a therapeutic target in GC.

Fig. 1. TMEM65 is upregulated in GC patients and predicts poor survival.

A TMEM65 mRNA expression was upregulated in GC tissues as compared with normal tissues in cohort I and TCGA cohort, and furtherly confirmed in paired GC tumor tissues from cohort I. B TMEM65 mRNA expression was upregulated in GC compared with paired adjacent normal tissues, as shown by regular RT-PCR, and TMEM65 protein expression was upregulated in GC tumor tissues than that in adjacent normal tissues as determined by IHC. C Kaplan–Meier survival analysis from cohort I showed GC patients with high TMEM65 expression had poorer survival. Kaplan–Meier survival analysis from TCGA cohort showed that GC patients with high TMEM65 expression (all stage, stage III-IV) had poorer survival compared with GC patients with low TMEM65 expression. D TCGA cohort showed a positive correlation between DNA copy number and TMEM65 mRNA expression level. E Kaplan–Meier survival analysis from cohort II showed GC patients with high TMEM65 DNA copy number had poorer survival than those with low TMEM65 DNA copy number.

Fig. 2. TMEM65 promotes GC cell growth, migration and invasion in vitro.

A The expression of TMEM65 in GC cell lines was determined by Western blot. B Ectopic expression of TMEM65 in AGS cells was confirmed by qRT-PCR and Western blot. C, D Ectopic expression of TMEM65 in AGS cells significantly promotes cell viability C and cell clonogenicity D. E Ectopic expression of TMEM65 in AGS cells obviously accelerated G1-S cell cycle progression, determined by flow cytometry. F Western blot showed overexpression of TMEM65 increased the protein expression of Cyclin-D1, CDK4, PCNA and reduced the expression of P53, P21^cip1. G Ectopic expression of TMEM65 in AGS cells inhibited early and late apoptosis, determined by flow cytometry. H Western blot showed ectopic expression of TMEM65 in AGS cells reduced the active expression levels of cleaved-caspase-3, cleaved-caspase-7, cleaved-caspase-8, cleaved-caspase-9, and cleaved-PARP. I Representative images of wound healing assay revealed that ectopic expression of TMEM65 promoted cell migration in AGS cells. J Representative images of Matrigel invasion transwell assay revealed that ectopic expression of TMEM65 promoted cell invasion in AGS cells. K Western blot showed ectopic expression of TMEM65 increased the protein expression of N-cadherin, β-catenin, and decreased the expression of E-cadherin.

Fig. 3. Knockdown of TMEM65 in GC cells suppresses cells growth, migration and invasion in vitro.

A Knockdown of TMEM65 by siRNA in MKN7 and MKN74 was confirmed by Western blot. B, C Knockdown of TMEM65 in MKN7 and MKN74 cells significantly inhibited cell viability B and colony formation ability C. D Knockdown of TMEM65 in MKN7 and MKN74 cells caused G1-S arrest which was determined by flow cytometry. E Knockdown of TMEM65 decreased the protein expression of Cyclin-D1, CDK4, PCNA and increased the expression of P53, P21^cip1. F Knockdown of TMEM65 in MKN7 and MKN74 cells promoted early and late apoptosis, determined by flow cytometry. G Western blot showed TMEM65 knockdown increased the expression level of cleaved-caspase-3, cleaved-caspase-7, cleaved-caspase-8, cleaved-caspase-9, and cleaved-PARP. H Knockdown of TMEM65 significantly inhibited migration ability in MKN7 and MKN74 cells. I Knockdown of TMEM65 significantly decreased cell invasion ability in MKN7 and MKN74 cells. J Western blot showed knockdown of TMEM65 decreased the protein expression of N-cadherin, β-catenin, and increased the expression of E-cadherin.

Fig. 4. Knockdown of TMEM65 inhibits tumor growth and migration in vivo and TMEM65 is a therapeutic target in GC.

A Knockdown of TMEM65 by shRNA in MKN74 was confirmed by Western blot, and stably knockdown of TMEM65 significantly inhibited cell viability. B Knockdown of TMEM65 in MKN74 cells significantly inhibited xenograft tumor growth and tumor weight of NOG mice, as shown by the growth curve of tumor volume at the experiment. C Knockdown efficiency of TMEM65 in xenografts was verified by IHC. D Knockdown of TMEM65 decreased the Ki67 positive cells percentage in subcutaneous xenografts, determined by IHC staining. E Knockdown of TMEM65 inhibited lung metastasis in nude mice. F Targeting depletion of TMEM65 by VNP-encapsulated TMEM65-siRNA significantly reduced tumor volume and tumor weight of nude mice, as shown by the growth curve of tumor volume and the tumor weight at the end of the experiment.

Fig. 5. TMEM65 activates PI3K/Akt/mTOR signaling and YWHAZ is the downstream target of TMEM65.

A RNA sequencing followed by KEGG pathways analysis revealed TMEM65 activated PI3K/Akt signaling. B Gene expression changes after overexpression of TMEM65 in PI3K/Akt signaling. C Ectopic expression of TMEM65 significantly reduced PI3K/Akt activity while knockdown of TMEM65 showed opposite effect. D Overexpression of TMEM65 enhanced the protein expression of PI3K/Akt/mTOR signaling related markers in GC cells, while knockdown of TMEM65 showed the opposite effect. E The inhibition efficiency on p-Akt of PI3K inhibitor GDC-0941 was confirmed by Western blot. F AGS cells treatment with PI3K inhibitor GDC-0941 (0.5 μmol/L) and Capivasertib (1 nmol/L), inhibited the promoting effect of TMEM65 on cell viability. G Workflow of downstream factor identification of TMEM65 by LC-MS. H Co-IP followed by Western blot analyses confirmed the binding between TMEM65 and YWHAZ in AGS cells. I TMEM65 and YWHAZ co-localized in cytoplasm of GC cells (AGS, MKN74 and MKN7) determined by Western blot. J TMEM65 and YWHAZ are mainly co-localized in cytoplasm in AGS and GC tissues as demonstrated by fluorescence microscope. K Ectopic expression of TMEM65 increased the stability of YWHAZ in AGS cells. L TMEM65 decreased YWHAZ ubiquitination and increased its protein level.

Fig. 6. Oncogenic function of TMEM65 is depended on YWHAZ in GC cells.

A YWHAZ was upregulated in GC tissues as compared with normal tissues from TCGA cohort. B Kaplan-Meier survival analysis from cohort I and TCGA cohort showed GC patients with high YWHAZ expression had poorer survival. C1 Knockdown of YWHAZ in MKN7 and MKN74 cells was confirmed by Western blot. C1–C3 Knockdown of YWHAZ in MKN7 and MKN74 cells significantly inhibited cell viability C1, colony formation ability C2 and PI3K/Akt signaling, evidenced by increased FHRE activity C3. D–H Knockdown of YWHAZ in AGS cells significantly reduced the promoting effect of TMEM65 on cell viability D, cell clonogenicity E, anti-apoptosis effect F, cell migration ability G, cell invasiveness H, and activation of PI3K/Akt signaling, determined by luciferase reporter assay I and Western blot J. K Schematic illustration of the molecular mechanism of TMEM65 in GC.