STOML2 Restricts Mitophagy and Increases Chemosensitivity in Pancreatic Cancer through Stabilizing PARL-induced PINK1 degradation

doi:10.21203/rs.3.rs-1952708/v1

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STOML2 Restricts Mitophagy and Increases Chemosensitivity in Pancreatic Cancer through Stabilizing PARL-induced PINK1 degradation

https://doi.org/10.21203/rs.3.rs-1952708/v1

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Pancreatic cancer remains one of the most lethal diseases with a relatively low 5-year survival rate, while gemcitabine-based chemoresistance happens constantly. Mitochondria, as the power factory in cancer cells, are involved in the process of chemoresistance. The dynamic balance of mitochondria is under the control of the mitophagy process. Stomatin-like protein 2 (STOML2) locates in the mitochondrial inner membrane and expresses highly in cancer cells. In this study, using a Tissue Microarray (TMA), we found that high STOML2 expression was correlated with higher survival of patients with pancreatic cancer. Meanwhile, proliferation and chemoresistance of pancreatic cancer cells could be retarded by STOML2. Besides, we found STOML2 was positively related to mitochondrial mass and negatively related to mitophagy in pancreatic cancer cells. STOML2 stabilized PARL and further prevented gemcitabine-induced PINK1-dependent mitophagy. We also performed subcutaneous xenografts to verify the enhancement of gemcitabine therapy induced by STOML2. These findings suggested that STOML2 regulated the mitophagy process through a PARL/PINK1 pathway, thereby reducing the chemoresistance of pancreatic cancer. STOML2-overexpression targeted therapy might be helpful for gemcitabine sensitization in the future.

STOML2

mitophagy

pancreatic cancer

gemcitabine

PINK1

Pancreatic ductal adenocarcinoma, generally referring to pancreatic cancer, remains one of the most lethal diseases. Compared with the obvious progress of survival benefits in many other cancer types, the current 5-year survival rate of pancreatic cancer is still about 11% (1). Gemcitabine (GEM)-based regimens remain to be the first-line chemotherapies. However, chemoresistance frequently happens and significantly threatens the long-term survival of pancreatic cancer patients. In a variety of chemoresistance mechanisms, metabolism alteration such as glycolysis has long been widely studied (2). Besides enhanced glycolysis in pancreatic cancer, some mitochondrial and key subcellular compartments in energy production, are also vital for pancreatic cancer progression by regulating tumor anabolism, calcium homeostasis and redox balance (3). Recently, more evidences have indicated that mitochondria participated in the process of chemoresistance (4).

Normal and healthy mitochondria are “power factories” in cancer cells, which serve as the location for the tricarboxylic acid cycle and generate adenosine triphosphate (ATP). The quantity and quality of mitochondria are under a dynamic quality control system named mitophagy, which could selectively remove dysfunctional mitochondria and maintain cell homeostasis (4). Mitophagy contains classical and non-classical pathways (5). The classical pathway is mainly induced by PINK1 (PTEN induced kinase 1) and PRKN (parkin RBR E3 ubiquitin protein ligase). Mechanistically, damaged mitochondria accumulate full-length PINK1 on its outer membrane, recruiting free PRKN in the cytoplasm and resulting in the followed aggregation of phosphorylated Ub chains on the mitochondrial outer membrane (6). There are many proteins that could influence the PINK1-PRKN pathway and affect mitophagy, including PARL (presenilin associated rhomboid like), a kind of intramembrane protease that locate at the mitochondrial inner membrane and could slice full-length PINK1 to cleaved-PINK1. Cleaved-PINK1 would be degraded by proteasome and loss its function of inducing mitophagy (7). Previous studies have suggested that the quantity and quality of mitochondria are closely connected with cell apoptosis, which might contribute to chemoresistance (4). Upon loss of transmembrane potential, damaged mitochondria released proapoptotic proteins such as cytochrome C to induce a caspase cascade. Besides, mitochondria serve as the appropriate site for aerobic glycolysis, glutamine metabolism and ROS metabolism, all of which have proved to exert a significant effect on chemoresistance (2, 4). Therefore, mitochondria play a vital role in pancreatic cancer chemoresistance which deserve further research.

Stomatin-like protein 2 (STOML2 or SLP2) was primarily identified as a protein located in the mitochondrial inner membrane, maintaining the stability of mitochondria (8, 9). Previous studies suggested that STOML2 promoted the progression of multiple malignant tumors including liver cancer (10), head and neck squamous cell carcinoma (11), ovarian cancer (12), colorectal cancer (13), and even pancreatic cancer (14). However, the relevant study in pancreatic cancer carried out by Chao et al. only demonstrated the relevance of STOML2 and pancreatic cancer patients’ prognosis, lacking the support of high-level clinical data such as tissue microarrays. The detailed molecular mechanism in this study was not very clear. Therefore, the function and mechanism of STOML2 in pancreatic cancer deserve further study. In this study, we found that in our cohort, high STOML2 expression was related to good prognosis and mainly located in the mitochondria, which could prevent mitophagy via stabilizing PARL and finally reduce chemoresistance to gemcitabine.

Bioinformatics analysis

GEPIA website containing TCGA and GTEx database was employed. In the TCGA database, samples with high STOML2 expression (above the mean) were allocated to the high STOML2 expression group, while samples with low STOML2 expression (below the mean) were allocated to the low STOML2 expression group. Log-rank test was utilized to analyze survival data of pancreatic cancer samples. Public proteogenomic data of pancreatic cancer tissue and Pearson correlation coefficients were employed to analyze the correlation between STOML2 and PARL in protein levels (15).

Tissue microarray analysis and immunohistochemistry (IHC)

Tissue microarray section (4 µm thick), including 79 pairs of pancreatic ductal adenocarcinoma samples and adjacent healthy pancreatic tissues were purchased from Shanghai Outdo Biotech CO., LTD. Enrolled patients have received little preoperative treatment in this study. For immunohistochemistry (IHC), the tissue microarray section was incubated with STOML2 monoclonal antibody (1:500 dilution, Proteintech, China) overnight at 4°C. The following IHC was performed according to the previously described protocol (16). The staining results were photoed by microscopy (Nikon ECLIPSETs2R). The results of staining were determined by two experienced pathologists independently. Staining score = Tumor cells proportion × staining intensity. Proportion: 0 (no stained tumor cells), 1 (< 10% stained tumor cells), 2 (10–25% stained tumor cells), 3 (26–49% stained tumor cells), and 4 (≥ 50% stained tumor cells). Intensity: 0 (negative particles), 1 (lightly yellow particles), 2 (brownish-yellow particles) and 3 (brown particles). The staining score between 0 to 5 was defined as the low STOML2 expression group, while ≥ 6 as the high expression group.

Cell lines and culture conditions

The pancreatic cancer cell lines PANC1 and BxPC-3 were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM (HyClone, Utah, USA) and RPMI-1640 (HyClone, Utah, USA) media respectively with 10% fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin & streptomycin (Biological Industries, Israel) and were grown in the cell incubator (37°C with 5% CO2).

Transfection assay and stable cell line construction

When seeded cells reached 70% confluence, they were infected with a STOML2-overexpression plasmid (STOML2 OE) or negative control (Vector) (Shanghai Genechem, China). STOML2 cDNA was subcloned into a pcDNA3.1(+) vector. Small interfering STOML2 RNA (siSTOML2) and negative control (NC) were purchased from RiboBio (Guangzhou, China) to construct STOML2 knockdown cells. The transfection process was performed by Lipofectamine 3000 (Invitrogen, Lithuania) according to the manufacturer’s instructions. To construct a stable STOML2-overexpression cell line, STOML2 (Homo sapiens) ORF-sequences were cloned into Ubi-MCS-3FLAG-SV40-Puro lentiviral vectors (Shanghai Genechem, China). The lentiviral vectors were utilized to package viral particles. PANC1 cells were infected with the lentivirus for over 24h. Then, puromycin (1µg/ml, Sigma-Aldrich, Missouri, USA) was added in the medium to remove uninfected cells.

Conditional culture

At 48h after transfection, the cells were cultured with the addition of gemcitabine (10µM for PANC1; 1µM for BxPC3, Vianex S.A.-Plant C, Greece), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10µM, MedChemExpress, USA) or chloroquine (10µM, MedChemExpress, USA) for 24h to analyze cell activities. Equal volumes of PBS or DMSO were used as control.

RNA isolation and qRT-PCR

TRIzol reagent (Invitrogen, California, USA) was utilized to extract total RNA. The quality of extracted RNA was analyzed by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) according to the 260-and 280-nm (A260/280) wavelengths. Complementary DNA was synthesized by a reverse transcription kit (ThermoFisher, Lithuania). Quantitative RT–PCR (qRT-PCR) was conducted with SYBR Green Master Mix (Applied Biosystems, Lithuania). β-actin was determined to be the internal control for mRNA detection. The relative expression of mRNAs was calculated via the 2^−ΔΔCT method. All primers used in this study are displayed in the Supplement Table 1.

Western blot and Co-immunoprecipitation (Co-IP)

Proteins in cells and tissue samples were extracted by RIPA lysis buffer with protease inhibitor (Beyotime, China). The extraction of cytosol and mitochondrial protein was performed by mitochondrial extraction kit (Solarbio, China) according to the manufacturer’s instructions. The concentrations of extracted protein were quantified by BCA Protein Assay Kit (Beyotime, China). The western blot experiment was performed according to the previously described protocols (17).

The first antibodies in this study: anti-STOML2 (10348-1-AP, Proteintech, China), anti-PARL (sc-514836, Santa Cruz, Texas, USA), anti-PINK1(sc-518052, Santa Cruz, Texas, USA), anti-LC3B (18725-1-AP, Proteintech, China), anti-Vinculin (66305-1-Ig, Proteintech, China), anti-Caspase-3 (sc-7272, Santa Cruz, Texas, USA), Anti-Cleaved Caspase-3 (ab2302, Abcam, UK), anti-COXIV (11242-1-AP, Proteintech, China), anti-α-Tubulin (11224-1-AP, Proteintech, China). The second antibodies: Goat anti-Rabbit IgG (ZSGB-BIO, ZB-2301, China), Goat anti-Mouse IgG (ZSGB-BIO, ZB-2305, China). For the co-immunoprecipitation (Co-IP) assay, all procedures were accomplished according to the manufacturer’s instructions (ThermoFisher, Massachusetts, USA). Briefly, PANC1 and BxPC3 cells were lysed by modified RIPA buffer. Cell lysates were mixed with protein A/G agarose beads-conjugated STOML2 antibody or IgG for 2h at room temperature on a rotating plate. Then, the following procedures were performed as Western blot with the first antibody and second antibody (ab131366, Abcam, UK).

Cell viability and colony formation assays

At 24h after transfection, PANC1 or BxPC3 cells were seeded in 96-well plates (3000 cells per well). Then, cell viability was measured at 0 h, 24 h, 48 h, 72 h and 96 h using Sulforhodamine B (SRB) assay (Sigma-Aldrich, Missouri, USA) (18). The optical density at the 564nm wavelength (OD540) was measured by a microplate reader. Besides, the CCK8 assay (Dojindo Molecular Technologies, Japan) was also employed to analyze cell viability (OD450-OD630). For colony formation assay, transfected cells were suspended at single-cell level and plated into 6-well plates (1000 cells per well). After 14 days, the cells were stained with 0.4% crystal violet (Sigma-Aldrich, Missouri, USA) with methanol. The plate was washed three times with water and photographed.

Growth inhibition assay with gemcitabine

For growth inhibition assays, pancreatic cancer cells were seeded into 96-well plates (3000 cells per well) at 24 h after transfection. After cell adherence (incubation for 4–6 h), gemcitabine (Vianex S.A.-Plant C, Greece) concentration gradients (1nM to 1mM for BxPC3; 10nM to 10mM for PANC1) were added into each well. SRB assay was performed after additional 48h incubation to determine the cell viability and inhibition rate.

Apoptosis assay

PANC1 and BxPC3 cells were seeded into 6-well plates. At 24h after transfection (siRNA and plasmids), cells were treated with gemcitabine (10µM for PANC1; 1µM for BxPC3, Vianex S.A.-Plant C, Greece). At 48 h after treatment, the cells were re-suspended in the binding buffer. Next, the cells were stained with propidium iodide (PI) and Annexin V-FITC based on the manufacturer’s instructions (Yishan Biotechnology, China). Followed analysis was carried out via FlowJo V10.8.1.

Quantitation of mitochondrial mass and reactive oxygen species (ROS)

Mitochondrial mass within PANC1 and BxPC3 was accessed by incubating cells with MitoTracker (Beyotime, China) for 30min at 37℃. Then, the cells were treated with DAPI (Beyotime, China) for 5min at 37℃. Fluorescent results about mitochondrial mass were taken photographs via confocal microscopy (AX, Nikon). The mitochondrial mass was finally analyzed via ImageJ V1.53. For measurement of intracellular ROS levels, ROS assay kit (Solarbio, China) was employed to measure the intracellular ROS levels of PANC1 and BxPC3. Briefly, transfected PANC1 and BxPC3 were treated with 10µM DCFH-DA in the serum-free medium for 20min at 37℃. For staining the nuclei, cells were further treated with Hoechst (Beyotime, China) for 5min at 37℃. Fluorescent results were taken through photographs via confocal microscopy (AX, Nikon). Fluorescent results about ROS were taken through photographs via microscopy (EVOS, ThermoFisher, Massachusetts, USA). The ROS levels were finally analyzed via ImageJ V1.53.

Mitophagy Detection

At 48h after transfection, PANC1 and BxPC3 cells were seeded into cell culture imaging slides (ThermoFisher, Massachusetts, USA) (5000 cells per chamber). After incubation for 24h, Mitophagy Detection Kit (Dojindo Molecular Technologies, Japan) containing mitophagy dye (red) and lysosome dye (green) was employed to assess mitophagy levels according to the manufacturer’s protocol. The co-localization between red and green represented the occurrence of mitophagy (yellow). Fluorescent images were taken via confocal microscopy (AX, Nikon). The mitophagy levels were finally analyzed via CellProfiler V4.2.1.

Animal experiments

All animal experiments in this study were performed under the guidelines of the Institutional Animal Care and Use Committee (Beijing, China). Control and STOML2 overexpressed PANC-1 cells were stably constructed via the aforementioned lentivirus. The cells were subcutaneously injected into the right back on 6-week-old female BALB/c nude mice (Chinese Academy of Sciences, China) (5× 10⁶ cells in 300 µl of PBS per mouse). Each group contained six mice. Tumor sizes were assessed using a caliper to measure the two perpendicular diameters of the xenografts (Tumor volume (mm³) = 1/2 ×length × width²). To confirm the role of STOML2 in promoting chemosensitivity in vivo, mice were administrated with gemcitabine through intraperitoneal injection (25 mg/kg, twice weekly) after the tumor volume reached 80mm³. After 30 days of administration, mice were euthanized. The xenografts were excised to perform further study in weight, morphology and immunohistochemistry.

Statistical analysis

All statistical results and graph representations were performed using the GraphPad Prism 9 Software. The differences among different groups were tested using the paired or unpaired Student’s t-test. Kaplan-Meier method was used to calculate the overall survival (OS) curves. P-value of < 0.05 (two-sided) was determined to be significant.

STOML2 was highly expressed in pancreatic cancer but related to longer survival

To assess the expression of STOML2 in pancreatic cancer and its relationship with overall survival, we analyzed STOML2 expression via the TCGA database. The expression of STOML2 in normal pancreatic tissues was also accessed from the GTEx database. Compared to the normal pancreatic tissue, STOML2 was highly expressed in pancreatic cancer (Fig. 1A). We further employed clinical samples and tested the STOML2 protein levels in pancreatic cancer tissues by a Tissue Microarray (TMA). The results supported that STOML2 was highly expressed in pancreatic cancer tissues when compared with their paired neighboring normal pancreas (Fig. 1B and C). Then, we compared STOML2 protein levels in human pancreatic cancer cell lines (PANC1, MIA PaCa-2, BxPC3) and normal pancreatic ductal epithelial cell line (HPNE). The expression of STOML2 protein in pancreatic cancer cells was higher than that in pancreatic ductal epithelial cells (Fig. 1). Next, we explored the correlations between STOML2 expression levels and clinicopathologic parameters among the 99 patients in the TMA. Compared to the male, STOML2 was highly expressed in the female group (p = 0.019; Table 1). Moreover, the association between STOML2 expression and overall survival (OS) time in pancreatic cancer patients was explored via Kaplan-Meier analysis. The results indicated that the patients with the low STOML2 expression group had significant shorter OS than high STOML2 expression group in both the TCGA database and TMA (Fig. 1D and E). Hence, STOML2 was remarkably high-expressed in pancreatic cancer which indicated a good prognosis in pancreatic cancer patients.

STOML2 inhibited pancreatic cancer cells proliferation and chemoresistance

STOML2 was inhibited by small interfering RNA (siSTOML2) and overexpressed by over-expression plasmid (STOML2 OE) in pancreatic cancer cell lines (PANC1 and BxPC3). The proliferation of pancreatic cancer cells was monitored by SRB assay, CCK8 assay and colony formation assay. These results showed that the cell proliferative ability was significantly decreased after STOML2 overexpression, while dramatically upregulated after STOML2 inhibition in PANC1 and BxPC3 (Fig. 2A-E). Considering the STOML2 expression has a strong relationship with the prognosis of pancreatic cancer patients, we further tested the function of STOML2 in gemcitabine resistance, which is the first-line chemotherapy against pancreatic cancer (19). In PANC1 and BxPC3, STOML2 inhibition could significantly reduce the inhibition rate of gemcitabine treatment in multiple concentrations, while STOML2 overexpression could upregulate the inhibition rate under gemcitabine treatment (Fig. 2F-I). Therefore, STOML2 could inhibit the proliferation and chemoresistance of pancreatic cancer cells.

STOML2 was primarily located in mitochondria and positively associated with mitochondrial mass in pancreatic cancer cells

According to the previous reports, STOML2 is a membrane-anchored protein located on the inner membrane of mitochondria (20). To further verify the location of STOML2 in pancreatic cancer cells, the cytoplasmic proteins (C) and mitochondrial proteins (M) were extracted from four pancreatic cancer cell lines (Mia-Paca-2, PATU8988T, PANC1, and BxPC3). Vinculin and COXIV were chosen to be the reference standard for the expression and purity of cytoplasmic and mitochondrial protein respectively (21, 22). The results suggested that STOML2 was primarily located in the mitochondria of pancreatic cancer cells (Fig. 2J). We next investigated the function of STOML2 in mitochondria. Mitotracker was utilized to label mitochondria. The results showed that STOML2 inhibition decreased mitochondrial mass while STOML2 overexpression increased mitochondrial mass in PANC1 (Fig. 2. L), which indicated that STOML2 might repress mitophagy and decrease gemcitabine resistance (23).

STOML2 restricted full-length PINK1 and mitophagy in pancreatic cancer cells

We further confirmed the relationship between STOML2 expression and PINK1-mediated mitophagy. There are many formations of PINK1 with different lengths, but only the full-length PINK1 (63KDa) could functionally recruit Parkin and induce the mitophagy process (24). Because the expression of full-length PINK1 was low in the general state (Fig. 3A), we employed carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10 µM) to elevate PINK1 levels, making it suitable to observe the effects of STOML2 on full-length PINK1 in pancreatic cancer cells (25). After downregulating STOML2 in PANC1 and BxPC3, the full-length PINK1 was upregulated (Fig. 3A and B), while over-expressing STOML2 could inhibit the full-length PINK1 levels in PANC1 and BxPC3 (Fig. 3C and D). Given that mitophagy is a part of autophagy, we further detected autophagy flux, which could partially reflect mitophagy levels (26). Chloroquine (10µM) treatment was used to block the autophagy flux, as reflected in the ratio of LC3BII/LC3BI (27). Downregulating STOML2 in both PANC1 and BxPC3 significantly increased the ratio of LC3BII/LC3BI (Fig. 3E and F), while upregulating STOML2 could decrease the ratio of LC3BII/LC3BI (Fig. 3. G and H). Therefore, these results suggested that STOML2 repressed the full-length PINK1 and the overall autophagy levels in the pancreatic cancer cell, which indicated the key role of STOML2 in mitophagy.

To further confirm the function of STOML2 in mitophagy, we utilized lysosome dye and mitophagy dye to detect mitophagy levels in PANC1 and BxPC3 (28). After inhibiting STOML2 in PANC1 and BxPC3, the intensity of mitophagy and the area of colocalization between lysosome and mitophagy was enhanced. Consistently, overexpressing STOML2 could decrease the intensity of mitophagy and the area of colocalization between lysosome and mitophagy (Fig. 3I-N). Therefore, STOML2 could effectively restrict the mitophagy of pancreatic cancer cells, which might mediate its role in reducing chemoresistance and longer OS of pancreatic cancer patients.

STOML2 alleviate gemcitabine-induced PINK1 elevation and mitophagy-mediated chemoresistance by stabilizing PARL

A previous study has proved that gemcitabine treatment could improve PINK1-induced mitophagy (29). To ensure the relationship between gemcitabine and mitophagy in pancreatic cancer cells, gemcitabine was added to the culture medium of PANC1 (10µM) and BxPC3 (1µM). The expression levels of PINK1 were detected after 48h. Gemcitabine treatment could obviously elevate the levels of full-length PINK1 (Fig.S1 A and B). The results suggested that gemcitabine could elevate the basic autophagy level in pancreatic cancer cells (Fig. S1 C and D). We next utilized lysosome dye and mitophagy dye to further detect mitophagy levels. Consistently, after gemcitabine administration, the mitophagy staining was enhanced and the colocalization area between lysosome and mitophagy was also significantly increased (Fig. S1 E and F). Hence, gemcitabine could promote the PINK1-induced mitophagy in pancreatic cancer cells.

To explore whether STOML2 repressed gemcitabine-induced PINK1 elevation and chemoresistance, we detect the full-length PINK1 under gemcitabine treatment after downregulating/upregulating STOML2. Compared to the control groups, downregulating STOML2 could further increase the full-length STOML2, while upregulating STOML2 dramatically reversed the elevation of full-length PINK1 caused by gemcitabine (Fig. 4A-D). Previous reports suggested STOML2 could directly stabilize PARL, a kind of protease that could degrade PINK1 (20). We thus tested the association between STOML2 and PARL. Based on the proteomic data of 140 pancreatic cancer cases (15), there is a significant positive association between STOML2 and PARL in protein levels (Fig. 4E, P = 0.02, R = 0.24). After altering the expression levels of STOML2, the expression of PARL is also changed along with the STOML2 in PANC1 and BxPC3 (Fig. 4F and G). Next, we detected the direct interaction between STOML2 and PARL by co-immunoprecipitation. In both PANC1 and BxPC3, the STOML2 antibody could also pull down PARL, which suggested that STOML2 could directly bind and stabilize PARL in pancreatic cancer cells (Fig. 4H and I). To certificate the STOML2/PARL/PINK1 pathway and its role in chemoresistance, rescue experiments were performed. After STOML2 overexpression, inhibiting PARL could reverse the downregulation of full-length PINK1 (Fig. 4G). The chemosensitivity phenotype driven by STOML2 overexpression was also reversed by PARL downregulation (Fig. 4K). Consistently, inhibiting PINK1 could reverse the chemoresistance phenotype under STOML2 downregulating background (Fig. 4. L and M). Hence, STOML2 repressed the mitophagy-mediated chemoresistance through STOML2/PARL/PINK1 pathway.

STOML2 decreased chemoresistance by promoting apoptosis of pancreatic cancer cells

To further explore the mechanism by which STOML2 inhibited chemoresistance, we examined the apoptosis levels of PANC1 and BxPC3 cells induced by gemcitabine treatment. Compared with the control groups, STOML2 downregulation could reduce the proportion of cleaved caspase3 to full-length caspase3, while STOML2 overexpression could increase the proportion of cleaved caspase3 to full-length caspase3 (Fig. 5A and B). Therefore, STOML2 promoted gemcitabine-induced apoptosis in protein levels. We further employed flow cytometry to confirm the role of STOML2 in cell apoptosis. Consistently, after downregulating STOML2, the apoptotic cells (including early and late apoptotic cells) were significantly decreased, while overexpressing STOML2 could obviously increase apoptotic cells (Fig. 5C-H). These results suggested that STOML2 could promote gemcitabine-induced apoptosis in pancreatic cancer cells.

Extensive cell mitophagy could result in functional mitochondria loss and cell apoptosis (30), however, PINK1-induced mitophagy could elevate the mitochondrial biogenesis pathway (31). Therefore, we detected the expression levels of key proteins in mitochondrial biogenesis, containing NRF1, TFAM, and PGC1α. Downregulating STOML2 in PANC1 and BxPC3 could upregulate the expression of PGC1α, NRF1, and TFAM in both RNA and protein levels (Fig. S2 A-D). Consistently, upregulating STOML2 in PANC1 and BxPC3 could downregulate the expression levels of NRF1, TFAM, and PGC1α (Fig. S2 E and F). Hence, STOML2 downregulation could also enhance the dynamic mitochondrial turnover, which could further prevent excessive mitophagy and maintain cancer cell survival. Recent evidence showed that accumulated damaged mitochondria with compromised mitophagy could generate amounts of ROS (32, 33). High levels of intracellular ROS could induce apoptosis (34). Hence, we measured ROS levels after downregulating or overexpressing STOML2 in PANC1 and BxPC3. Downregulating STOML2 could significantly reduce the ROS levels and upregulating STOML2 could dramatically increase the ROS levels (Fig.S3). These results suggested that pancreatic cancer cells with high STOML2 expression levels preferentially have compromised mitophagy, accumulated damaged mitochondria and higher intracellular ROS levels, which might contribute to the gemcitabine-induced apoptosis.

STOML2 decreased chemoresistance of pancreatic cancer in vivo

To validate the function of STOML2 in decreasing chemoresistance in vivo, we employed nude mice and stable STOML2-overexpression PANC1 cells to construct subcutaneous xenograft. After the volume of subcutaneous xenograft reached 80mm³, the qualified mice were injected with gemcitabine and the tumor volumes were measured every three days (Fig. 6A). 4 weeks after gemcitabine administration, the involved mice were sacrificed (Fig. 6B). The tumor volume and tumor weight were directly compared between the two groups (Fig. 6C and D). These results showed that STOML2 obviously decreased tumor growth under gemcitabine treatment in vivo. Thus, STOML2 could effectively reduce chemoresistance of pancreatic cancer in vivo. The STOML2 expression and downstream PARL were further verified in vivo (Fig. 6E and F), indicating the stable STOML2 overexpression and STOML2/PARL/PINK1 pathway in vivo.

Pancreatic cancer remains one of the most lethal diseases at present. Despite decades of research, chemotherapy resistance remains a thorny issue limiting the survival time of pancreatic cancer patients. There are many mechanisms by which pancreatic cancer cells induce chemoresistance (35). Recently, accumulated studies suggested that the mass and quality of mitochondria are vital for pancreatic cancer cells survival, which also contribute to the dynamic adaptation for adverse environmental conditions, such as starvation, hypoxia and chemotherapy treatment (4). Mitochondria are not only major energy providers, but also the site of the tricarboxylic acid cycle, amino acid and lipid metabolism (2). As an organelle, the status of individual mitochondria is highly different. Mitophagy is an essential process for the removal of damaged mitochondria and plays an important role in many physiological and pathological processes. However, mitophagy plays a dual and complex role in carcinogenesis and cancer progression (33, 36). In this study, we reported that STOML2 was highly expressed in pancreatic tumor tissue but related to longer survival, which might support the idea that mitophagy could repress tumorigenesis but promote cancer progression (37, 38). The underlying mechanism was also explored in this study. STOML2 could directly stabilize PARL, which could degrade PINK1 and inhibit its role in inducing mitophagy. Simultaneously, STOML2 inhibited autophagy flux in pancreatic cancer, which might contribute to the chemoresistance in pancreatic cancer (39). Consequently, ROS levels and apoptosis were significantly increased in pancreatic cancer cells, ultimately leading to reduced chemoresistance and prolonged survival.

For chemoresistance, previous studies suggested that the impaired mitophagy restricted the metabolic plasticity, which made the pancreatic cancer cells susceptible to metformin treatment (40). However, another related research showed that mitophagy could inhibit malignant behaviors including chemoresistance in pancreatic cancer cells (41). Therefore, there remain many controversies between mitophagy and chemoresistance in pancreatic cancer, which deserves further study (42). Dysfunctional mitochondria are the main source of ROS (43, 44). After production, overload ROS directly changed mitochondrial membrane structure, contributing to the leakage of ROS from mitochondria. As a result, relatively high levels of ROS damaged the nuclear DNA and even cause strand breaks, finally promoting genomic instability (45). During cancer progression, ROS is also a double-edged sword. Some reports showed that certain levels of ROS could stimulate cancer proliferation and metastasis, which is partially mediated by inactivating tumor suppressor phosphatases. Whereas high levels of ROS are cytotoxic, resulting in apoptosis (46). Therefore, STOML2 might extensively inhibit the clearance of damaged mitochondria, which led to the boots of intracellular ROS levels and followed apoptosis. Mitophagy and its related ROS elimination remain unclear in pancreatic cancer and deserve further study (47).

However, when the intense mitophagy over a certain level, too few mitochondria failed to supply enough energy and metabolites for cell survival, resulting in cell death. Therefore, in addition to mitophagy, mitochondrial biogenesis is also necessary for maintaining healthy mitochondrial mass and respiratory capacity, assisting cancer cell survival and metastasis (48). There are also multiple regulators participate mitochondrial biogenesis. Previous study suggested that mitophagy could promote the translocation of transcription factor EB (TFEB) to the nucleus, which induced the transcription of PGC1α mRNA (49). PGC1α is identified as a key factor (50) to activate nuclear respiratory factor (NRF1) and then increase the expression of nuclear mitochondrial transcription factor gene (TFAM) (51). In this study, we found that mitophagy driven by low STOML2 could promote the expression levels of PGC1α, NRF1, and TFAM, which indicated that low STOML2-promoted mitophagy could also induce mitochondria biogenesis. The young mitochondria could provide sufficient energy but produce lower ROS, which might further promote cell survival and chemoresistance.

There are other related studies that also reported the role of STOML2 in cancer. In hepatocellular cancer, STOML2 could directly bind with PINK1 and promote its followed mitophagy (10), which is extremely different from our present study in pancreatic cancer. The huge difference in gene mutation and expression between hepatocellular cancer cells and pancreatic cancer cells might contribute to the distinct role of STOML2 in mitophagy. While another report showed that STOML2 could upregulate hexosamine biosynthetic pathway and promote pancreatic cancer metastasis (14). However, the function of STOML2 in mitophagy and chemoresistance was not detected in that study. Protein might contribute to different phenotypes via distinct molecular pathways. This study also has some shortcomings. First, we only analyzed STOML2 overexpression pancreatic cancer cells in vivo, which lacked the data from the STOML2 downregulation group. Second, the peptide detailed sequence of STOML2 that mediates the direct contact between PARL and PINK1 should be addressed, which might provide more evidence for the STOML2/PARL pathway. In conclusion, our study found that STOML2 could repress PINK1-induced mitophagy by directly stabilizing PARL, thus inhibiting pancreatic cancer chemoresistance. Therefore, overexpression STOML2 might be a novel anticancer strategy in further pancreatic cancer treatment.

GEM, gemcitabine; ATP, adenosine triphosphate; PINK1, PTEN induced kinase 1; PRKN, parkin RBR E3 ubiquitin protein ligase; PARL, presenilin associated rhomboid like; STOML2, Stomatin-like protein 2; IHC, immunohistochemistry; ATCC, Afrmerican Type Culture Collection; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; SRB, Sulforhodamine B; LC3, microtubule-associated protein 1 light chain 3; CQ, chloroquine;

Ethics approval and consent to participate: All animal care and experiments were conducted following the guidelines for the Institutional Animal Care and Use Committee. Written informed consent was obtained from all patients for the use of resected tissues.

Acknowledgements: Not applicable

Author Contributions: Weibin Wang conceived, designed, and directed the study. Cheng Qin and Yuanyang Wang performed all experiments, data analyses and wrote the original manuscript. Bangbo Zhao, Zeru Li, Tianyu Li, Xiaoying Yang, and Yutong Zhao wrote and critically reviewed the manuscript.

Conflict of Interest: The authors have declared that no conflict of interest exists.

Data Availability Statement：Not applicable

Funding：Weibin Wang received the support from the National Natural Science Foundation of China (No.81773215; No.82173074) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2021-I2M-1-002).

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Supplementaryfigure1.tif
Supplementary Figure 1. GEM promoted PINK1-induced mitophagy in pancreatic cancer cells. (A-B) Immunoblot showed the protein levels of PINK1 and β-actin in PANC1 and BxPC3 after treating with GEM or control in cell medium for 24h. (C-D) Immunoblot showed the protein levels of LC3BI, LC3BII, and β-actin in PANC1 and BxPC3, which were treated with GEM, chloroquine (CQ, 10μM) or control. The corresponding ratios of LC3BII/LC3BI were calculated by the OD value of the corresponding blot band determined by ImageJ. (E) Fluorescent staining showed the colocalization between lysosome and mitophagy (mitochondria) in PANC1 and BxPC3 after treating GEM or control. The corresponding quantitative results were calculated by CellProfiler V4.2.1. *: P < 0.05; **: P<0.01.
supplementaryfigure2.tif
Supplementary Figure 2. STOML2 repressed mitochondrial biogenesis in pancreatic cancer cells. (A-B) Immunoblot showed the protein levels of PGC1α, NRF1, TFAM, STOML2, and Vinculin in PANC1 and BxPC3 after downregulating STOML2, which were treated with CCCP for 24h. (C-D) qRT-PCR showed the mRNA levels of PGC1α, NRF1 and TFAM, compared to β-actin in PANC1 and BxPC3 after downregulating STOML2. (E-F) Immunoblot showed the protein levels of PGC1α, NRF1, TFAM, STOML2 and Vinculin in PANC1 and BxPC3 after upregulating STOML2, which were treated with CCCP for 24h. *: P < 0.05; **: P < 0.01; ***: P < 0.001.
Supplementaryfigure3.tif
Supplementary Figure 3. STOML2 increased ROS levels in pancreatic cancer cells. (A-B) Fluorescent staining showed the ROS levels in PANC1 and BxPC3 after downregulating or upregulating STOML2. (C-F) Corresponding quantitative results of the ratios of ROS levels/cell number were calculated by ImageJ. **: P < 0.01; ***: P < 0.001.

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You are reading this latest preprint version

STOML2 Restricts Mitophagy and Increases Chemosensitivity in Pancreatic Cancer through Stabilizing PARL-induced PINK1 degradation

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Version 1

Abstract

Figures

Introduction

Materials And Method

Bioinformatics analysis

Tissue microarray analysis and immunohistochemistry (IHC)

Cell lines and culture conditions

Transfection assay and stable cell line construction

Conditional culture

RNA isolation and qRT-PCR

Western blot and Co-immunoprecipitation (Co-IP)

Cell viability and colony formation assays

Growth inhibition assay with gemcitabine

Apoptosis assay

Quantitation of mitochondrial mass and reactive oxygen species (ROS)

Mitophagy Detection

Animal experiments

Statistical analysis

Results

STOML2 was highly expressed in pancreatic cancer but related to longer survival

STOML2 inhibited pancreatic cancer cells proliferation and chemoresistance

STOML2 was primarily located in mitochondria and positively associated with mitochondrial mass in pancreatic cancer cells

STOML2 restricted full-length PINK1 and mitophagy in pancreatic cancer cells

STOML2 alleviate gemcitabine-induced PINK1 elevation and mitophagy-mediated chemoresistance by stabilizing PARL

STOML2 decreased chemoresistance by promoting apoptosis of pancreatic cancer cells

STOML2 decreased chemoresistance of pancreatic cancer in vivo

Discussion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1