Lysosomal TMEM9‐LAMTOR4‐controlled mTOR signaling integrity is required for mammary tumorigenesis

Dear Editor, Accumulating evidence suggests that dysregulated lysosomal membrane proteins, including vacuolar ATPase (vATPase) and themammalian target of rapamycin (mTOR), are involved in tumorigenesis [1]. Therefore, lysosomal proteins were proposed as potential therapeutic targets in cancer [1]. As one of the lysosome-related signaling pathways, mTOR signaling regulates cell proliferation, survival, motility, andmetabolism [2]. SincemTOR signaling activation promotes tumorigenesis, mTOR inhibitors (mTORi), AZD8055 [3], MLN0128 [4], and Rapalink-1 (the latest third-generation mTORi) [4], have been applied to several cancers. However, the limitations of mTORi include drug resistance and the lack of biomarkers. Recently, we identified transmembrane protein 9 (TMEM9) as a positive regulator of Wnt/β-catenin signaling in colorectal [5] and hepatocellular cancers [6]. TMEM9 facilitated the assembly of v-ATPase, which induced vesicular acidification and subsequent hyperactivation of β-catenin-mediated gene expression [5]. Given the vital role of TMEM9 in vesicular acidification and lysosomal dysfunction, we sought to determine the impact of TMEM9-dysregulated lysosomes on tumorigenesis. Detailed methods can be found in the Supplementary file. To assess the pathologic relevance of TMEM9 to cancer, we first analyzed the TMEM9 gene alteration and expression. Among human cancers, TMEM9 gene amplification and upregulation were frequently observed in breast can-

Next, we determined in vivo roles of TMEM9 in mammary tumorigenesis using a mammary-specific polyomavirus middle T antigen overexpression (MMTV-PyMT) mouse model. We confirmed that Tmem9 was highly expressed in mammary tumors of MMTV-PyMT mice (Supplementary Figure S3A Figure S3J). These data suggest that TMEM9 plays an oncogenic role in mammary tumorigenesis.
Next, we sought to dissect the mechanism by which TMEM9 contributes to mammary tumorigenesis. Since TMEM9 binds to and activates v-ATPase for lysosomal vesicular acidification [5,6], we examined the impact of TMEM9 on lysosomal proteins. We isolated the lysosomes  Table S3). Intriguingly, among the top 20 lysosomal proteins differentially detected, LAMTOR4, a component of the LAMTOR complex, became undetectable in TMEM9-KD cells ( Figure 1F; Supplementary  Table S4). Next, we examined the effect of TMEM9 on lysosomal LAMTOR4 by immunofluorescence staining for LAMTOR4 and lysosomal associated membrane protein 1 (LAMP1), a lysosome marker, in TMEM9-KD ZR-75-1 cells (Supplementary Figure S4E-F). Co-localization analysis showed the reduced co-localization between LAM-TOR4 (green) and LAMP1 (red) in TMEM9-KD cells (Supplementary Figure S4G). The scatter dot plot also exhibited decreased co-localization of LAMTOR4 with LAMP1 by TMEM9 KD ( Figure 1G). Consistently, MMTV-PyMT:Tmem9-KO tumors also showed a decrease in colocalization between LAMTOR4 and LAMP1, compared to MMTV-PyMT:Tmem9-WT tumors ( Figure 1G; Supplementary Figure S4H-I). These results suggest that TMEM9 is required for the lysosomal localization of LAMTOR4. The lysosomal v-ATPase-Ragulator complex activates the mTORC1 signaling [7]. As a member of the Ragulator complex, LAMTOR4 modulates Ragulator assembly and contributes to anchoring mTOR to the lysosomal membrane [7]. Therefore, decreased levels of LAMTOR4 in the lysosome by TMEM9 KD may disrupt mTOR sig-naling activity. Indeed, mTOR and mTOR downstream effector phosphorylated proline-rich Akt substrate of 40 kDa (p-PRAS40) were downregulated in Tmem9-KO mammary tumors ( Figure Figure S5G-H).
To further determine the impact of TMEM9-regulated mTOR signaling on cell proliferation, we performed a rescue experiment by lentiviral overexpression of wild-type or constitutively active mutant of mTOR (L1460P) [8] in TMEM9-KD cells. mTOR L1460P rescued the cell proliferation inhibition induced by TMEM9 KD, while mTOR WT failed to do ( Figure 1J). These results suggest that TMEM9activated mTOR signaling is required for breast cancer cell proliferation.
Although several mTORi have been introduced for cancer therapy, mTORi resistance is a recurring issue [9]. To overcome mTORi resistance, combinatorial therapies have been used [10]. Given the TMEM9 gene amplification in breast cancer and TMEM9-activated mTOR signaling, it is plausible that mTORi resistance might be due to TMEM9 upregulation in breast cancer. It should be noted that mTOR inhibition did not upregulate TMEM9 expression (Supplementary Figure S6A). To determine whether the TMEM9-v-ATPase axis can be a therapeutic target to overcome mTORi resistance, we treated breast cancer cells with v-ATPase inhibitors (bafilomycin A1 and concanamycin A) in combination with mTORi (rapamycin and Rapalink-1). It should be noted that TMEM9 high breast cancer cells (MDA-MB-453) were more sensitive to v-ATPase inhibitors than TMEM9 low breast cancer cells (BT-474) (Supplementary Figure S6B). We assessed the impact of pharmacological blockade of the TMEM9-v-ATPase axis on mTORi response. The bliss synergy scoring analysis showed that rapamycin or Rapalink-1 combined with bafilomycin A1 or concanamycin A synergistically inhibited breast cancer cell viability across multiple concentrations ( Figure Figure S6E). These results suggest that the pharmacological blockade of the TMEM9v-ATPase axis sensitizes tumor cells to mTORi.
Herein our results identified a new role of the TMEM9v-ATPase axis in modulating mTOR signaling via LAM-TOR4. In breast cancer cells harboring the TMEM9 gene amplification, TMEM9-activated v-ATPase aberrantly regulates lysosome-associated signaling, including the mTOR pathway. Upon TMEM9 upregulation, TMEM9-induced v-ATPase assembly and activation likely stabilize the LAMTOR4-containing Ragulator complex assembly in the lysosomal membrane, contributing to mTOR signaling hyperactivation ( Figure 1L-M). The detailed molecular mechanisms of mTOR downregulation and mislocalization of LAMTOR4 by TMEM9 depletion need to be elucidated in future studies. The efficacy of further combinatorial therapies remains to be tested using in vivo preclinical models.
Together, our results unveiled that the lysosomal TMEM9-v-ATPase-Ragulator-Rag axis is indispensable for mTOR signaling integrity and breast cancer cell proliferation and that TMEM9 may serve as a biomarker and a molecular target, which may overcome mTORi resistance and offer a viable therapeutic strategy for TMEM9-associated breast cancer. Chen, and Jae-Il Park analyzed the data. Shengzhe Zhang and Jae-Il Park wrote the manuscript. 3