Myotubularin-related phosphatase 3 promotes invasion and metastasis by repressing autophagy of colorectal cancer cells

doi:10.21203/rs.3.rs-1457715/v1

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Myotubularin-related phosphatase 3 promotes invasion and metastasis by repressing autophagy of colorectal cancer cells

https://doi.org/10.21203/rs.3.rs-1457715/v1

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posted 28 Mar, 2022

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With the development of modern society, human living standards have improved substantiallythus altering their lifestyles, particularly eating habits. These changes haveresulted in an increased incidence of colorectal cancer (CRC) in recent years. More than one million new cases of CRC are diagnosed worldwide and more than half a million deaths occur due to CRC, each year, making CRC the main cause of cancer-related mortality. In this study, we explored the role of myosin-related phosphatase 3 (MTMR3) in the invasion and metastasis of CRC by transfecting the human CRC cell line, HCT116 cells, with lentivirus-mediated short interfering RNA. A wound healing assay was performed to evaluate the effect of MTMR3 knockdown on cell migration ability. The effect of MTMR3 knockdown on the endothelial-mesenchymal transition and cell apoptosis was evaluated by western blot analysis. Our results indicate that knockdown MTMR3 significantly reduced the invasion and metastatic ability of HCT116 cells and promoted cell autophagy (p < 0.05).These results demonstrate that silencing of MTMR3 shows potential as an effective treatment for CRC.

Colorectal cancer

MTMR3

Bafilomycin A1

autophagy

cell migration

According to the latest surveillance epidemiological results of the National Cancer Institute, 45 of every 100,000 patients are diagnosed with colorectal cancer (CRC) each year, and 16.4 of 100,000 patients die each year from CRC (1), making CRC the main cause of cancer-related mortality worldwide. In recent years, researchers have explored the effect of healthy colonic epithelial cells on CRC and have made good progress; however, the overall prognosis of CRC remainsunsatisfactory(2, 3).

Myotubularin-related phosphatase 3 (MTMR3) is a phosphoinositide (PI) phosphatase belonging to the myotubularin (MTM) family and is directed against phosphatidylinositol (3)-phosphate (PtdIns3P) and phosphatidylinositol PI 3-phosphatase of alcohol (3, 5)-bisphosphate. MTMR3 contains a PH-GRAM domain at its N-terminal, which is necessary for its binding to PI lipids. MTMR3 can hydrolyze PtdIns3P and PtdIns(3, 5)P2 in vitro(4, 5).

MTMR3 is a widely expressed myosin, whichwhen overexpressed, localizes in the cytoplasm and reticulum; however, its specific role remains unclear. Previous studies showed that MTMR3 regulates local PtdIns3P levels and negatively regulates autophagy. Knockdown of MTMR3 increases the formation of autophagosomes, whereas overexpression of wild-type MTMR3 causes autophagosomes to become significantly smaller and autophagy activity to decreased (6). Yoo et al. reported that MTMR3 can negatively regulate the growth of lung cancer cells (7). Their results indicated that MTMR3 increases the expression of p27, a cyclin-dependent kinase inhibitor, and stops the cell cycle at G1 phase. Recently, researchers revealed the novel role of MTMR3 in oral cancer. Kuo et al. found that MiR-99a exerted anti-metastatic activity by inhibiting MTMR3 expression, making MTMR3 a potential therapeutic target for oral cancer treatment (8). In our previous study, we showed that MTMR3 promotes the growth of CRC cells and that MTMR3 knockdown can lead to a decline in cell proliferation and colony formation of CRC cells(9). However, the functional role of MTMR3 in CRC are unclear and further studies are urgently required.

In the present study, we explored the role of MTMR3 expression in CRC cell migration and autophagy and demonstrated that MTMR3 silencing led to decreased cell migration and increased autophagy.

Cell culture

The human CRC cell line HCT116 and human embryonic kidney cell line 293T were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). HCT116 cells were cultured in McCoy's 5A medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum. DMEM (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum was used to culture 293T cells. The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

Construction of MTMR3 shRNA lentiviral vector and cell infection

The sequence of Lv-shMTMR3-S1 was 5′-GGATGCCATCATTGCCCTT-3′ and that of Lv-shMTMR3-S2 was 5′-GCATGTAACTTCAAGGTTT-3′. The sequence was designed based on the human MTMR3 gene (NM_021090.3) and cloned into the PLKO.1 vector (Addgene, Watertown, MA, USA). Non-silencing short interfering RNA was used as a control;its sequence being 5′-TTCTCCGAACGTGTCACGT-3′. DNA sequencing was conducted to confirm the production of lentiviral-based short hairpin RNA (shRNA)-expressing vectors. Lentiviruses were generated by transfection of 293T cells at 80% confluence with a modified PLKO.1 vector and packing plasmids psPAX2 and pMD2G (Addgene). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer's instructions. At 48 h after transfection, the supernatant was collected and lentiviral particles were harvested by ultracentrifugation (4000 ×g) at 4°C for 10 min, and then filtered through a 45-µm filter. For lentivirus infection, HCT116 cells (5 × 10⁴ cells/well) were seeded into 6-well plates and transduced with MTMR3 shRNA (Lv-shMTMR3-S1 and Lv-shMTMR3-S2) or control shRNA (Lv-shCon) expressing lentivirus at a multiplicity of infection of 15 and 50, respectively. Infection efficiency was determined by measuring the number of red fluorescence protein-expressing cells under a fluorescence microscope at 72 h after infection. More than 80% of the cells expressed red fluorescent protein, indicating that transfection was successful.

Western blotting

After infection for 5 days, HCT116 cells were washed with cold phosphate-buffered saline and lysed in 2X sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris-HCl (pH 6.8), 10 mM EDTA, 4% SDS, and 10% glycine) for 1 h at 4°C. After centrifugation (12,000 ×g for 15 min), protein content was measured with an enhanced BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts (30 µg) of protein in each lane were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked and then incubated with primary antibodies: rabbit anti-MTMR3 (1: 1000, number 12443), mouse anti-E-cadherin (1: 1000, number 14472), rabbit anti-N-cadherin (1: 1000, number 13116), rabbit anti-Snail (1: 1000, number 3839), rabbit anti-Slug (1: 1000, number 9585), vimentin (1: 1000, number 5741), rabbit anti-β-catenin (1: 1000, number 8480), rabbit anti-LC3 (1: 1000, number 12741), rabbit anti-P62 (1: 1000, number 16177), rabbit anti-S6K (1: 1000, number 2708) (the above antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA), and mouse anti-GAPDH (1: 60,000, number Sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), overnight at 4°C. After washing with Tris-buffered saline containing Tween 20, the blots were incubated with horseradish peroxidase-labeled anti-rabbit (1: 5000, number Sc-2054, Santa Cruz Biotechnology) or anti-mouse (1: 5000, number Sc-2005, Santa Cruz Biotechnology) secondary antibody for 2 h at room temperature and then visualized by adding super ECL detection reagent (Applygen, Beijing, China). To further evaluate the effect of MTMR3 on the apoptosis of HCT116 cells, the cells were co-treated with Bafilomycin A1 (Baf A1), a lysosomal activity inhibitor(10).

Wound healing assay

HCT116 cells transfected with the corresponding vectors were seeded into 6-well plates to form a single confluent cell layer. Wounds were made with 100-µL tips in the confluent cell layer. At 0 and 24 h after wound scratching, the width of the wound was photographed with a phase-contrast microscope.

Transwell migration and invasion assays

The Transwell migration assay was conducted as follows: cultured cells were pre-incubated with Mitomycin-C (10 µg/mL) for 1 h at 37°C to suppress proliferation,following which 2×10⁴ cells in serum-free medium containing 0.1% bovine serum albumin were placed in the upper chamber of the insert and 500 µL complete medium was added to the bottom chamber. After 12 h of incubation, the cells were washed with PBS, and cells adhered to the lower membrane of the inserts were counted after staining with 0.1% crystal violet. The numbers of cells were counted under an inverted microscope. The Transwell invasion assay was conducted as described above but the upper chamber was coated with Matrigel (5 mg/mL, BD Biosciences, Franklin Lakes, NJ, USA) instead. These experiments were also performed in triplicate.

Immunofluorescence assay

HCT116 cells were fixed on sterile sheet glass and disposed of with melatonin for 48 h. Following this, the cells were immersed in 4% (w/v) paraformaldehyde for 20 min in phosphate-buffered saline after permeabilization with 0.5% Triton X-100 for 10 min. The cells were blocked in 10% bovine serum albumin for 1 h and then incubated with a rabbit primary antibody (1:200, Abcam, Cambridge, UK) overnight. On the next day, a fluorescein-conjugated goat anti-rabbit secondary antibody (1:100, Boyun, Shanghai, China) was added to the glass slides and left for 1 h at 37°C. The cells were then counterstained with DAPI (Beyotime) and visualized using an ECLIPSE Ni microscope (NIKON, Tokyo, Japan).

Statistical analysis

All results represent the mean ± standard deviation from three independent experiments. Student's t-test was used to evaluate the differences between groups, using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Differences were considered significantwhenP < 0.05.

Effective knockdown of MTMR3 by shRNA in colon cancer cells

To study the role of MTMR3 in CRC, we first prepared a recombinant lentivirus vector of MTMR3 and performed cell infection. HCT116 was successfully infected with Lv-shMTMR3-S1, Lv-shMTMR3-S2, or Lv-shCon, and a blank group was used as a control. The gene silencing effects of MTMR3 shRNAs were evaluated by western blotting analysis. As shown in Figure S1, both Lv-shMTMR3-S1 and Lv-shMTMR3-S2 significantly downregulated the expression of MTMR3 compared to that in the control group. Moreover, S2 showed a better interference effect than did S1.

MTMR3 knockdown led to decreased cell migration and invasion

After confirming the knockdown efficiency of the shRNA targeting MTMR3, the effect of low MTMR3 expression on cell migration was evaluated in a wound healing assay. As shown in Fig. 1, the migration and invasion rates of HCT116 cells significantly decreased after Lv-shMTMR3 infection compared to those in the control group.

MTMR3 knockdown led to decreased epithelial mesenchymal transition (EMT)

EMT is a critical process in CRC metastasis. To further explore the role of MTMR3 in CRC progression, the protein expression levels of E-cadherin, β-catenin, Snail, N-cadherin, Slug, and vimentin were measured by western blot analysis (Fig. 2). The expression of E-cadherin and β-catenin was increased significantly after MTMR3 knockdown compared to those in control samples. Additionally, the mesenchymal makers Snail, N-cadherin, Slug, and vimentin were significantly inhibited after knockdown of MTMR3. Our results indicate that MTMR3 plays an important role in CRC metastasis.

MTMR3 knockdown led to increased autophagy

We then measured the effect of MTMR3 suppression on the degree of protein degradation during autophagy of CRC cells. In cells in which MTMR3 was suppressed, the expression of autophagy-dependent protein degradation markers were significantly increased (LC3II/LC3I and P62), as shown in Fig. 3A. Moreover, MTMR3 knockdown down-regulated the protein expression of p-S6K (Fig. 3B). The results of immunofluorescence analysis also showed that LC3 was up-regulated in HCT116 cells after MTMR3 knockdown (Fig. 3C).

After treatment with Baf A1, the expression of P62 and Snail increased, whereas the expression of E-cadherin decreased, indicating that autophagy flow was reduced (Fig. 4).

MTMR3 promoted cell migration and invasion by downregulating autophagy

To further determine how MTMR3 regulates the migration of CRC cells, rescue experiments combined with a wound healing assay were conducted. Our results showed that MTMR3 knockdown significantly decreased the migration ability of CRC cells (Fig. 5). However, after treatment with Baf A1, the decreased HCT116 cell migration ability was reversed, indicating that MTMR3 promoted cell migration by downregulating autophagy.

Given the recent changes in human lifestyle, especially those concerning diet, the incidence of CRC has increased in recent years in Asia, despite improved treatments for controlling cancer progression (11, 12). As one of the main causes of cancer deaths, nearly one million new cases of CRC have been diagnosed worldwide, with half a million deaths each year. Therefore, the need for new cancer treatment methods is urgent. Among the various new cancer treatment methods, gene therapy, based on the molecular function of cancer cell survival, has received increased attention. In this study, we focused on identifying an oncogenic target in CRC and investigating the effects of silencing this gene on CRC cell migration.

We observed a novel role for MTMR3 in CRC. MTMR3 showed diverse effects on the proliferation of cultured cells, which were distinct from those described in previous studies of different cell types(13, 14). Therefore, it is critical to further understand the regulatory activity of MTMR3 on CRC proliferation and evaluate the role of this molecule in CRC. Unlimited proliferation and division are two important characteristics of tumor cells. In this study, we found that when two specific MTMR3 shRNAs suppressed MTMR3 expression in HCT116 cells, the cell migration ability of HTC116 cells significantly reduced, suggesting that MTMR3 is a potential target for CRC treatment.

CRC cell metastasis first begins when a cancer cell leaves the primary cancer. This process is typically accompanied by changes in the epithelial cadherin (E-cadherin), immunoglobulin (Ig) superfamily, selectin, and integrin.EMT is thought to play an important role in tumor invasion and metastasis (12). EMT refers to the phenomenon in which epithelial cells lose their differentiated phenotype and acquire certain mesenchymal phenotypes in a specific microenvironment (15, 16). The occurrence of EMT is an extremely complicated process. Many intracellular and extracellular signaling molecules, protein molecules, and such, participate in its regulation and maintain the morphology of interstitial cells.EMT-producing cancer cells often exhibit increased protein expression, such as Snail, Twist, and ZebWait. Activation of the transforming growth factor (TGF)-β pathway can lead to direct phosphorylation of Smad, following which P-Smad can interact with other transcription factorsto regulate the expression of Snail, Twist, and Zeb-related families, thereby down-regulating the protein levels of epidermal biomarkers such as E-cadherin and up-regulating interstitial biomarkers to promoteEMT (17). Studies have shown that Smad4 can inhibit cell invasion and change SW480 cells from a mesenchymal-like phenotype to a non-polar epithelial phenotype. Moreover, overexpression of Smad4 is accompanied by down-regulation of endogenous TGF-β (18). In contrast, deletion of Smad4 leads to abnormal activation of STAT3, which may directly lead to Zeb1 overexpression and EMT (19). Deletion of Smad4 has been observed in at least 30% of metastatic CRC cases, which is accompanied by upregulation of E-cadherin and β-catenin (15, 16, 20). Additionally, TGF-β can induce apoptosis, and cells must inhibit death caused by TGF-β. Studies have shown that up-regulation of Snail not only promotes EMT, but also up-regulates Akt and Bcl-xL, thereby inhibiting TGF-β-induced apoptosis (21). However, while suppressing apoptosis, Snail also affects the cell cycle. It can down-regulate cyclin D2 and cause cell cycle arrest (22). In the tumor microenvironment, Snail can be regulated by multiple pathways, such as by activation of HIF1, HIF2, and Notch pathways in hypoxic environments, as well as nuclear factor-κB and TGF-β during inflammation (23). These series of studies showed that the Snail pathway plays an important role in EMT. In the present study, we found that EMT-related proteins are regulated by MTMR3. After knocking down MTMR3, E-cadherin and β-catenin were increased significantly, whereas the mesenchymal makers Snail, N-cadherin, Slug, and vimentin were significantly inhibited, indicating that MTMR3 regulates the EMT of CRC cells.

However, the role of EMT in tumor metastasis is controversial, mainly because the phenotypes caused by transient and reversible EMT processes are not observed in vivo. EMT is generally considered as an important step in the process of cancer cells leaving the tumor in situ and metastasizing to other sites to form metastases. However, some studies suggested that EMT is not required for cancer cell metastasis. In addition, the protein markers that indicate activation of the EMT conversion pathway have been observed in many other unrelated cellular processes, such as apoptosis, supporting that EMT is not required for metastasis (24). Fischer et al. (25) found that EMT was not necessary for tumor metastasis by Cre-mediated fluorescent labeling of interstitial cells to establish a mouse model of lung cancer with spontaneous lung metastasis. The study showed that in primary epithelial tumors, only a small proportion of tumor cells underwent EMT. Non-EMT tumor cells retained their epithelial cell characteristics. In addition, EMT was inhibited by overexpression of miR-200, which did not affect the development of lung metastases, indicating that EMT was not the main force promoting the development of lung metastases in breast cancer. Therefore, the role of MTMR3 in CRC metastases requires further analysis.

Autophagy controls a wide range of physiological processes, such as starvation, cell differentiation, cell survival, and cell death through the renewal of long-lived proteins, processing of damaged organelles and misfolded proteins, and renewal of cellular components after nutritional deprivation (26). Autophagy has a dual role as a survival mechanism and cell death mechanism. Emerging evidence suggest that autophagy and apoptosis can coexist or occur sequentially to induce cell death (27, 28). In this study, we found that when two specific MTMR3 shRNAs inhibited MTMR3 expression in HCT116 cells, autophagysignificantly increased. Baf A1 is a macrolide antibiotic isolated from Streptomyces species and inhibits H⁺-ATPase in vacuoles (29). Baf A1 has been reported to have strong biological activities, including inhibition of cell growth, differentiation, and apoptosis, as well as anti-inflammatory and anti-tumor properties (30). At high doses (0.1–1 mM), Baf A1 is often used as an inhibitor to block autophagosome and lysosomal fusion or to inhibit lysosomal activity, which is a key step in autophagy at later stages (31). In our study, we found that Baf A1 reversed reduced cell migration caused by MTMR3 knockdown. These results confirm that MTMR3 increases the cell migration capacity of CRC cells by inhibiting autophagy.

We foundin our study that MTMR3 plays an important role in colon cancer cell migration and autophagy. Silencing of MTMR3 suppressed cell migration by promoting autophagy and blocking the EMT of CRC cells. Thus, strategies targeting MTMR3 in CRC may be of clinical value.

Baf A1 Bafilomycin A1

CRC Colorectal cancer

EMT Epithelial-mesenchymal transition

MTMR3 Myotubularin-related phosphatase 3

PI Phosphoinositide

PtdIns3P Phosphatidylinositol (3)-phosphate

PtdIns(3,5)P2 Phosphatidylinositol (3,5)-biphosphate

shRNA Short hairpin RNA

siRNA Short interfering RNA

TGF Transforming growth factor

Not applicable.

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the Natural Science Foundation of Zhejiang Province (LY15H030014), the Funding Project of Health and Family Planning Commission of Zhejiang Province (2018KY217), the Funding Project Administration of Traditional Chinese Medicine of Zhejiang Province (2018ZA009), and the Funding Project of CSCO, Chinese Society of Clinical Oncology (Y-MX2016-047).

Availability of data and materials

Not applicable.

Authors’ contributions

Bo’an Zheng, Weiding Wu, Xiaodong Xu and Yuanming Jing performed the experiments and the scientific literature search. Xinye Hu, Ziang Wan and Weike Zhou contributed to the figures and the writing of the manuscript. Jungang Zhang, Xiaoyan Jia and Bingchen Chen took part in the experiment planning. Jungang Zhang, Xiaoyan participated in the data analysis. All authors reviewed the manuscript. Rui Chai conceived and designed the study and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

Figures1.docx
Figure S1. (A) Protein levels of myosin-related phosphatase 3 (MTMR3) in HCT116 cells following 4 treatments (Lv-shMTMR3-S1, Lv-shMTMR3-S2, Lv-shCon, and Con) measured by western blotting. (B) Densitometric analysis of western blottingis shown. The experiment was repeated three times. P< 0.01, *P< 0.001 vs. Con.
Figures2.docx
Figure S2. The original image of western blot in Figure 4.

PDF

Version 1

posted 28 Mar, 2022

You are reading this latest preprint version

Myotubularin-related phosphatase 3 promotes invasion and metastasis by repressing autophagy of colorectal cancer cells

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell culture

Construction of MTMR3 shRNA lentiviral vector and cell infection

Western blotting

Wound healing assay

Transwell migration and invasion assays

Immunofluorescence assay

Statistical analysis

Results

Effective knockdown of MTMR3 by shRNA in colon cancer cells

MTMR3 knockdown led to decreased cell migration and invasion

MTMR3 knockdown led to decreased epithelial mesenchymal transition (EMT)

MTMR3 knockdown led to increased autophagy

MTMR3 promoted cell migration and invasion by downregulating autophagy

Discussion

Conclusions

Abbreviations

Declarations

References

Competing Interests

Supplementary Files

Status:

Version 1