The mitochondrial protein YME1 Like 1 is important for non-small cell lung cancer cell growth

The expression and biological function of the mitochondrial inner membrane protease YME1L (YME1 Like 1 ATPase) in NSCLC are tested here. Bioinformatical analyses and results from local human tissues show that YME1L expression is elevated in NSCLC tissues. YME1L upregulation was observed in primary and immortalized NSCLC cells. In NSCLC cells, shRNA-mediated silence of YME1L or dCas9/sgRNA-induced knockout (KO) of YME1L robustly suppressed cell growth and migration, and provoking apoptosis. YME1L shRNA/KO resulted in mitochondrial dysfunctions in NSCLC cells, leading to mitochondrial depolarization, ROS accumulation and ATP depletion. Conversely, ectopic YME1L overexpression augmented NSCLC cell proliferation and motility. Akt-S6K1 phosphorylation was reduced after YME1L shRNA/KO in primary NSCLC cells, but augmented after YME1L overexpression. Importantly, YME1L KO-caused anti-NSCLC cell activity was attenuated by a constitutively-activate Akt1 (S473D) construct. In vivo, subcutaneous NSCLC xenograft growth was remarkably slowed following intratumoral YME1L shRNA AAV injection in nude mice. YME1L knockdown, Akt-mTOR inactivation and ATP reduction were detected in YME1L-silenced NSCLC xenografts. Taken together, overexpressed YME1L in NSCLC exerts pro-tumorigenic function.


Cells
A549 cells were reported previously [30,31] and were cultivated under RPMI medium plus serum. The primary NSCLC cells derived from three writteninformed consent patients (pNSCLC-1, pNSCLC-2 and pNSCLC-3), the primary lung epithelial cells-derived from two written-informed consent donors were described early [30,31], and cells cultured in medium described [30,31]. The Ethics Committee of Nanchang University approved the the protocols of the present study.

Human tissues
Fifteen (15) fresh NSCLC tissues and the matched adjacent normal epithelial tissues were from primary written-informed consent NSCLC patients (stage-III-IV) in authors' institutions. Tissue slides were tested via immunohistochemistry (IHC) staining using the described protocols [32].

YME1L shRNA or overexpression
The lentivirus encoding YME1L shRNA ((5'-GATCCCCGTGGCAGAGGAATTCATATTTCA AGAGAATATGAGTTCCTCTGCCACTTTTTGGAA A-3') or YME1L cDNA were provided by Dr. Cao [28] and were added to the described cells. After 48h, cells were maintained under puromycin-containing medium for another four passages. YME1L silencing or overexpression was verified at mRNA and protein levels. For xenograft studies, YME1L shRNA sequence/shC sequence was inserted into an AAV (adeno-associated virus) vector [28,29]. The construct and the AAV envelope plasmids were co-transfected to HEK-293 cells to generate shRNA-expressing AAV.

Thiobarbituric acid reactive substance (TBAR) assaying of lipid peroxidation
Tissue lysates, at 20 proteins per sample, were measured using a commercial TBAR kit (Cayman Chemical, MI) specifically quantifying lipid peroxidation and malondialdehyde (MDA) contents colorimetrically. TBAR intensity was examined at 555 nm with the reference of 590 nm.
Other cell functional assays and gene/protein expression/interaction detection NSCLC cells/epithelial cells with the designated YEM1L genetic treatment were seeded at optimized confluence and cultivated. CCK-8 cell viability, colony formation, the nuclear EdU/DAPI staining assay of cell proliferation, the caspase-3 activity assay, cell apoptosis detection by nuclear TUNEL/Hoechst 33342 staining, in vitro cell migration "Transwell" assays, JC-1 staining of mitochondrial depolarization were described in detail in our previous studies [30,31]. Trypan blue staining of cell death and ssDNA (single strand DNA) ELISA (Merck, Shanghai, China) were described in a previous study [35], with the Histone-bound DNA ELISA assays described in another study [36]. Quantitative real time-PCR (qPCR), Western blotting and co-immunoprecipitation (Co-IP) was described early as well [30,31]. CellROX staining of ROS content, tissue/cellular ATP contents and the mitochondrial complex I activity were measured using the described protocols [29,37]. Figure S1 listed the uncropped blotting images.

Statistical analysis
The detailed procedures of statistical analyses were reported in our previous studies [30,31]. All in vitro experiments were repeated five times. Error bars were mean ± standard deviation (SD).

YME1L expression is elevated in NSCLC
TCGA-LUAD database reveals that expression of YME1L is significantly elevated in NSCLC tissues ("Tumor", Figure 1A). The relatively low YME1L expression was detected in lung tissues ("Normal", Figure 1A). In NSCLC tumor tissues YME1L transcripts' number is robustly higher than its number in the adjacent normal tissues ( Figure 1B). The mRNA sequencing data from a total of 515 NSCLC-LUAD patients in TCGA were analyzed using the LinkedOmics functional module. Figure 1C, the volcano plot, shows red dot genes that were positively correlated with YME1L, whereas green dot genes were negatively correlated with YME1L (false discovery rate/FDR < 0.01). The top fifty genes positively correlating with YME1L expression were presented in a heat map ( Figure 1D). Significant KEGG term annotation by overrepresentation enrichment analysis (ORA) showed the top ten pathways enriched by YME1L-co-expressing genes ( Figure 1E). Many of these pathways are vital for cancer progression, including DNA replication, mismatch repair, cell cycle progression and citrate cycle (TCA cycle) ( Figure 1E). These bioinformatics studies show that overexpressed YME1L might exert a tumorigenic role in NSCLC.

YME1L expression is elevated in local NSCLC
YME1L expression was measured in local surgery-resection NSCLC tissues. NSCLC tissues ("T") and matched adjacent normal tissues ("N") were obtained from fifteen (n = 15) primary NSCLC patients (LUAD, stage III-IV). Analyzing tissue lysates confirmed that YME1L mRNA levels in NSCLC tumor tissues were dramatically higher than those in normal tissues (Figure 2A). Moreover, increased protein expression of YME1L was observed in NSCLC tissues of five patients ("T1" to "T5") ( Figure 2B). Quantified results combining YME1L protein blotting data of the 15 sets of tissues demonstrated that the protein expression of YME1L was significantly elevated in NSCLC tissues ( Figure 2B, the right panel). The tissue IHC images further supported the protein upregulation of YME1L in NSCLC tissues ("T1" and "T2") of "Patient-1# and Patient-2#" (Figure 2C). The protein expression of YME1L in normal lung epithelial tissues was low ( Figure 2C).
In the primary pNSCLC-1, pNSCLC-2 and pNSCLC-3 cells [30,31] and the immortalized A549 cells, YME1L mRNA expression was higher than its expression in "pEpi1" and "pEpi2" primary lung epithelial cells (Figure 2D). The protein expression of YME1L was upregulated in the NSCLC cells ( Figure  2E and F), and low expression detected in the lung epithelial cells (Figure 2E and F). Thus, elevated YME1L expression is detected in local NSCLC tissues and NSCLC cells.
Next, to pNSCLC-2/3 cells and immortalized A549 cells, the lentivirus with YME1L shRNA was added and stable "shYME1L" cells were formed following selection. Expression of YME1L mRNA was indeed robustly decreased in the shYME1L NSCLC cells (Figure 3G). Silence of YME1L in the NSCLC cells decreased viability (Figure 3H), arrested cell proliferation ( Figure 3I) and hindered in vitro cell migration ( Figure 3J).

In pNSCLC-2/3 cells and A549 cells, YME1L
shRNA-induced silence of YME1L augmented Caspase-3 activity ( Figure 4G) and increased TUNEL nuclei ratio (Figure 4H and I). Moreover, YME1L silencing provoked cell death and increased Trypan blue staining in the NSCLC cells ( Figure 4J). Whereas in pEpi1 and pEpi2 lung epithelial cells, YME1L knockdown by YME1L shRNA (see Figure 3) failed to provoke apoptosis (Figure 4K and L). pNSCLC-1cells with the described genetic modification of YME1L or with control treatment were cultivated, the caspase-3 activity was tested (A), and listed proteins measured (B); The histone-DNA contents were tested (C). Cell apoptosis was tested via the nuclear TUNEL staining assay (D and E), and cell death measured by the Trypan blue staining assay (F). pNSCLC-2/3 cells, A549 cells, or pEpi1/2 epithelial cells, stably expressing YME1L shRNA ("shYME1L") or control shRNA ("shC") were cultured, the caspase-3 activity was measured (G and K), with cell apoptosis measured via the TUNEL staining assays (H, I and L). Cell death was measured as well (J). * P < 0.05 versus "Pare"/ "shC" cells. Scale bar=100 μm.
In pNSCLC-2/3 cells and A549 cells, YME1L shRNA also induced ROS production and increased the CellROX red fluorescence intensity ( Figure 5G). Moreover, depolarization of mitochondria, reflected by green monomer JC-1 accumulation, was detected in YME1L-silenced primary and A549 NSCLC cells (Figure 5H). In addition, ATP depletion was detected in the NSCLC cells with YME1L silencing (Figure 5I). Thus, YME1L depletion impaired mitochondrial functions in NSCLC cells.
Signaling changes in the pNSCLC-1 xenografts were examined. Specifically at Day-14 and Day-28, one pNSCLC-1 xenograft from the shYME1L-AAV group and the shC-AAV group was carefully isolated. Part of the fresh tumor xenografts were cut into five pieces and signalings were tested. YME1L expression was remarkably decreased in shYME1L-AAV pNSCLC-1 xenograft tissues (Figure 8E and F), where Akt (Ser-473) and S6K1 phosphorylation was robustly decreased (Figure 8G and H). Total Akt1 and S6K1 was again unaffected by shYME1L-AAV (Figure 8G and H). Moreover, ATP reduction was detected in shYME1L-AAV xenografts ( Figure 8I). Lipid peroxidation, or increased TBAR activity, was detected in YME1L-silenced pNSCLC-1 xenograft tissues ( Figure 8J). Srinivasainagendra et al., reported that in human colorectal cancer YME1L could be frequently mutated, and its mutation also occurring in other human cancers to a less degree [51]. YME1L inhibition led to significant death of cancer cells [23,52]. Silence of YME1L caused accumulation of Ndufb6, ND1, and Cox4, thereby suppressing cell proliferation [23].

Discussion
Recent studies have proposed a possible tumorigenic role of YME1L. Liu et al., have shown that overexpressed YME1L is important for orthotopic glioma xenograft growth in mice [28]. The same group further reported that TIMM44, another mitochondrial protein, promoted glioma cell growth possibly by increasing YME1L transcription and expression [29]. Liao et al., have implied that YME1L could be a promising biomarker for diagnosis and prognosis prediction in ovarian cancer [53]. YME1L is upregulated in ovarian cancer and is associated with worse overall survival [53]. Moreover, YME1L and it co-expressing genes are enriched in immune-related signaling pathways, supporting a possible inhibitory role of YEM1L in cancer immunotherapy [53]. Kakehashi et al., reported that expression of YME1L, together with other cytoskeletal proteins involved in endoplasmic reticulum stresses and mitochondrial dysfunctions, are overexpressed in HCV-associated hepatocellular carcinomas (HCC) [54]. Increased mitochondrial respiration and ATP generation are extremely important for NSCLC tumorigenesis and progression. Here we found that the mitochondrial protein YME1L exerted tumorigenic activity in NSCLC. TCGA database and local human tissues/cells results demonstrated that YME1L expression is elevated in NSCLC tissues and cells. YME1L shRNA or KO potently suppressed NSCLC cell viability, proliferation and in vitro migration, and provoking apoptosis. In addition, YME1L depletion caused mitochondrial dysfunctions, leading to depolarization of mitochondria, oxidative injury, DNA breaks and ATP depletion in different NSCLC cells. In vivo, the growth of subcutaneous primary NSCLC xenografts was hindered following YME1L shRNA AAV injection in nude mice. ATP reduction and oxidative injury were observed in YME1L-depleted xenograft tissues.
YME1L was recently shown to promote Akt activation [28]. Here activation of Akt-mTOR was reduced after YME1L shRNA/KO in primary NSCLC cells. It was augmented after ectopic YME1L overexpression. Our results supported that YME1L should be important for the integrity of mTORC1/2 and YME1L depletion disrupted the assembling of mTORC1/2. Importantly, YME1L KO-mediated anti-NSCLC cell activities, including proliferation arrest, migration inhibition, and apoptosis, were largely ameliorated by caAkt1. Akt-mTOR inhibition was observed in YME1L-silenced NSCLC xenograft tissues. Thus, Akt-mTOR activation is important for YME1L-promoted NSCLC cell growth.

Conclusion
The mitochondrial protein YME1L protein is overexpressed in NSCLC and exerts significant pro-tumorigenic activity possibly by supporting mitochondrial function and promoting Akt-mTOR activation.