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Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells Jie-qiong Peng a, b, Shu-mei Han b, Ze-hao Chen e, Jing Yang b, Yan-qing Pei b, Cong Bao d, Lei Qiao c, Wen-qiang Chen c, *, Bo Liu b, ** a
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China Department of Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China c The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, 107 Wenhuaxi Road, 250012, Jinan, China d Department of Pathology, Pingyi County People’s Hospital, Linyi, Shandong, 273300, China e Shandong First Medical University, Taian, Shandong, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2019 Accepted 13 November 2019 Available online xxx
Chaperone-mediated autophagy (CMA) is one of the three types of autophagy. In recent years, CMA has been shown to be associated with the pathogenesis of several types of cancer. However, whether CMA is involved in the pathogenesis of colorectal cancer (CRC) remains unclear. In this study, we investigated CMA activity in tissue specimens from CRC patients and mouse models of colitis-associated CRC (induced by administration of AOM plus DSS). In addition, we down-regulated CMA in CT26 colon carcinoma cells stably transfected with a vector expressing a siRNA targeting LAMP-2A, the limiting component in the CMA pathway, to explore the role of CMA in these cells. Apoptosis was detected using TUNEL assay, and the apoptosis-related proteins were detected using western blotting. Cell proliferation was assessed using MTT assay, Ki-67 labelling and western blotting for PCNA. We found that LAMP-2A expression was significantly increased in CRC patients and mouse models and varied according to the stage of the disease. Inhibition of CMA in CT26 cells facilitated apoptosis, as evidenced by increased TUNEL immunolabeling, increased expression of Bax and Bnip3, and decreased expression of Bcl-2. Cell proliferation assays showed that inhibition of CMA impeded the proliferation of CT26 cells. These data support the hypothesis that CMA is up-regulated in CRC, and inhibition of CMA may be a new therapeutic strategy for CRC patients. © 2019 Elsevier Inc. All rights reserved.
Keywords: Colorectal cancer Chaperone-mediated autophagy Apoptosis Proliferation
1. Introduction Chaperone-mediated autophagy (CMA) is an autophagic pathway in mammalian cells. In contrast to macroautophagy,
* Corresponding author. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, No. 107, Wen Hua Xi Road, Jinan, Shandong, 250012, China. ** Corresponding author. Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, 440 Jiyan road, 250117, Jinan, China. E-mail addresses: [email protected] (W.-q. Chen), 15553115688@163. com (B. Liu).
another form of autophagy, CMA is more specific, and only proteins that contain a specific targeting motif are degraded [1,2]. In recent years, CMA has been associated with the pathogenesis of several types of cancer, and increasing evidence has shown that CMA activity is increased in different cancer cell lines and various cancers of different origins [2e4]. CMA was also shown to be essential for the proliferation of human lung cancer cells [4] and gastric cancer cells [5]. Furthermore, inhibition of CMA in xenografts led to tumor shrinkage and reduced metastasis in mice [4]. Although autophagy has been well studied, most investigations have been focused on macroautophagy, and several studies have highlighted the controversial role of autophagy in cancer [6,7]. Some recent studies have shed light on this debate by examining the involvement of a less-well-studied type of autophagy, CMA [8].
https://doi.org/10.1016/j.bbrc.2019.11.081 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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However, there are few studies on the role of CMA in cancer, and only lung [4] and gastric cancer [5] have been well studied. Therefore, more research is needed. Colorectal cancer (CRC) is a major cancer with a substantial public health burden. It is the third most common diagnosed cancer in males and the second most common in females [9]. Despite recent advances in diagnosis and treatment, the survival rates for advanced stage CRC remain poor. Therefore, it is of great importance to clarify the underlying mechanisms of CRC and identify novel target molecules. Previous studies have shown that the CMA marker LAMP-2A (L2A) is upregulated in human CRC [5], indicating that CMA might play an important role. To test this hypothesis, we first verified CMA levels in both human specimens collected at different stages and CRC mouse models. In addition, we utilized a lentivirus-associated virus-mediated RNA interference (RNAi) technique to knock down the expression of LAMP-2A, the most important component of the CMA pathway, and investigated the effect of CMA dysfunction on cell proliferation and apoptosis using cultured mouse CT26 cells. 2. Materials and methods 2.1. Ethics statement All animal experimental protocols were approved by the Shandong Cancer Hospital and Institute, Shandong First Medical University, and Shandong Academy of Medical Sciences. Tissues from 45 patients with different stages of colorectal cancer, 15 patients with colorectal adenoma, and 15 patients with colitis were collected at Shandong Cancer Hospital, from 2016 to 2018. This study was approved by the Ethics Committee of the Shandong Cancer Hospital and Institute (SDTHEC20171209). All patients provided written informed consent before any experimental procedures were performed. 2.2. Animals Six-week-old male BALB/c mice (n ¼ 25) were obtained from Charles River Laboratories (Beijing, China) and were housed under a 12 h light/dark cycle. Fifteen of these mice were used to generate models of colitis-associated CRC [10,11]. Briefly, mice were administered AOM (10.0 mg/kg body weight; Sigma-Aldrich) as a single intraperitoneal injection and three subsequent cycles of 2.5% (wt/vol) DSS (molecular weight 36,000e50,000; MP Biomedicals) in drinking water for 7 days each. 2.3. Immunohistochemistry and immunofluorescence At 15 weeks, the mice were sacrificed, and the colorectum was harvested and fixed in 4% paraformaldehyde (PFA). These specimens were frozen in OCT and routinely sectioned into 6 mm-thick slabs for histological staining and immunofluorescence. Tissue sections were stained with hematoxylin-eosin (H&E), and frozen cross sections were immunostained using a rabbit polyclonal antibody against LAMP-2A (ab18528; Abcam) and a FITC-labeled secondary goat anti-rabbit IgG antibody (ZSJB-BIO, China). IHC staining was amplified with peroxidase-conjugated streptavidin complexes (ZSJB-BIO), and peroxidase was detected using AEC (ZSJB-BIO) substrate. The signals were visualized under a Manual Positive Fluorescence Microscope (Y-TV55; Nikon, Japan). 2.4. Cell lines and transfection CT26 colon carcinoma cells were obtained from ATCC and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotics at 37 C
in a humidified atmosphere of 5% CO2. LAMP-2A was silenced in CT26 cells with a lentiviral vector-mediated small interfering RNA (siRNA) obtained from Shanghai Genechem Co., Ltd. The sequences targeted by the siRNAs (si1, si2, and si3) were 50 -GACTGCAGTGCAGATGAAG-30 , 50 -CTGCAATCTGATTGATTA-30 , and 50 -TAAACACTGCTTGACCACC-30 , which are located in exon 8a of LAMP-2A and correspond to bases 1198e1216, 1331e1359, and 1678e1700, respectively [12]. Based on our previous experience, we performed two consecutive transfections at a 48 h interval, and the assays were carried out 48 h after the second transfection.
2.5. Western blotting CT26 cells were washed with cold PBS and homogenized on ice in RIPA lysis buffer (Beyotime, China) supplemented with complete protease inhibitor (Beyotime). Supernatants were collected after centrifugation at 12,000g for 30 min. The protein concentration in the supernatant was determined using the BCA Protein Assay Kit (Beijing Solarbio Science & Technology co., Ltd.). Protein samples were boiled with Laemmli sample buffer for 10 min, separated using 12% SDS-PAGE, and transferred to PVDF membranes (0.22 mm; Bio-Rad). After the membranes were blocked with 5% skim milk in TBST for 1 h, they were incubated overnight at 4 C with primary antibodies against LAMP-2A (Invitrogen), LAMP-2B (ab18529; Abcam), LAMP-2 (ab13524; Abcam), Bnip3 (ab109362; Abcam), Bcl-2 (ab182858; Abcam), and Bax (ab32503; Abcam). The protein levels in the western blots were normalized to b-actin (#4970, CST). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with a Luminescent Image Analyzer (Amersham Imager 600; GE, USA). The intensities of the corresponding protein bands were evaluated by densitometry using Image J. Protein expression was assessed relative to b-actin or other indicated protein.
2.6. Cell proliferation Cell proliferation was tested with MTT kit (Sigma) according to the manufacturer’s instruction and Ki-67 labelling. Slides containing CT26 cells were washed with PBS three times and fixed in PFA for 20 min. The cells of the slides were permeabilized by incubation with 0.3% Triton X-100 for 10 min at room temperature and then incubated with 5% goat serum for 30 min to block nonspecific binding. Next, the slides were incubated with an anti-Ki-67 primary antibody (ab15580; Abcam) for 12 h at 4 C, and then with a FITClabeled secondary goat anti-rabbit IgG antibody (ZSJB-BIO, China) on for 1 h. Nuclei were stained with DAPI, and cell fluorescence was analyzed using fluorescence microscopy (DS-Ri2; Nikon, Tokyo, Japan). The proliferation index was obtained by calculating the ratio of Ki-67-positive cells to the total cells in 10 fields of view. The expression of another pro-proliferation protein, PCNA (ab18197; Abcam), was analyzed using western blotting.
2.7. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The results are expressed as the mean ± SD of three independent experiments. Student’s t-test or one-way ANOVA analysis was used to compare the different treatment groups. Statistical significance was set at P < 0.05.
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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3. Results 3.1. Chaperone-mediated autophagy activity is increased in human colorectal cancer samples of different stages LAMP-2A is a key protein in CMA, and its level is the most direct indicator of CMA [13,14]. To investigate CMA levels in human CRC samples of different stages, we performed LAMP-2A immunostaining in tissue samples from patients with colitis and stage IeIII CRC who were diagnosed between 2016 and 2018. The results showed that LAMP-2A expression was significantly correlated with tumor staging (Fig. 1). LAMP-2A was almost undetectable in tissues from colitis patients, was slightly increased in tissues from patients with colorectal adenoma and Stage I CRC, but was significantly higher in tissue samples from patients with Stage II and III CRC (Fig. 1). These results were consistent with previous studies and further verified that human CRC tissues show elevated CMA activity. 3.2. Chaperone-mediated autophagy activity is increased in mouse colorectal cancer tissues To explore the variation in CMA in CRC, we evaluated the LAMP2A levels in mouse models of colitis-associated CRC (Fig. 2A and B). Immunofluorescence analysis of mouse colorectal tissues showed that LAMP-2A expression was high in the CRC tissues but was not observed in the tumor-adjacent tissues (Fig. 2C). Western blotting also showed a significant increase in the LAMP-2A levels in the CRC tissues compared to the levels in tumor-adjacent tissues (Fig. 2D and E). In addition, we also investigated the levels of another LAMP2 isoform, LAMP-2B. Analysis showed that the LAMP-2B levels were very low in both the CRC and tumor-adjacent tissues (Fig. S1). These results, along with the data from the human specimens described above, validated the importance of CMA in CRC and demonstrate that CMA is activated in CRC. 3.3. Blocking chaperone-mediated autophagy enhanced apoptosis in CT26 colon carcinoma cells To better illustrate the role of CMA in colon carcinoma cells, we inhibited CMA by knocking down LAMP-2A expression in CT26 cells using a vector-mediated RNA interference (RNAi) technique. As LAMP-2 undergoes alternative splicing of exon 8, leading to the isoforms LAMP-2A, B, and C, and only the LAMP-2A isoform is exclusively involved in CMA [14,15], we choose RNA sequences that specifically target the LAMP-2A exon [12]. All three of the LAMP-
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2A-specific siRNAs (referred to as si1, si2, and si3) downregulated LAMP-2A expression, although si2 and si3 showed the strongest down-regulation (Fig. 3A and B). LAMP-2B was not changed (Fig. 3A and C) and LAMP-2 was reduced in si2 and si3transfected cells (Fig. 3A and D). Therefore, si3 was used in subsequent experiments. Apoptosis can affect the occurrence and development of tumors, and it is of great significance in the treatment of tumors. Therefore, we investigated the role of CMA in apoptosis induced by TNF-a in CT26 cells. TUNEL assays showed that blocking CMA promoted tumor cell apoptosis, as TUNEL immunolabeling was increased in the si3-transfected cells compared to that in siNC-transfected cells (CTR) (Fig. 3E and F). Both pro-apoptotic (Bax and Bnip3) and antiapoptotic (Bcl-2) proteins were detected using western blotting in control siRNA-transfected (CTR) and si3-transfected cells after TNFa treatment. As shown in Fig. 3GeJ, blocking CMA increased the expression levels of Bax and Bnip3 and decreased those of Bcl-2. These data suggested that the inhibition of CMA enhanced the apoptosis of CT26 colon carcinoma cells. As a previous study showed that there was an intersection between two types of autophagy [16], we also checked the protein levels of the specific macroautophagy markers Beclin1, LC3B, and Atg5 to determine if autophagy initiation and progression were activated. None of these proteins were changed following LAMP-2A knockdown (Fig. S2), which confirms that LAMP-2A is not required for macroautophagy. 3.4. Chaperone-mediated autophagy is required for the proliferation of CT26 colon carcinoma cells As we observed that knock down of LAMP-2A in CT26 cells reduced cell proliferation rates compared to those in siNCtransfected cells, we analyzed cell proliferation using the MTT assay and Ki-67 labelling to validate this result and quantify cell proliferation. The MTT results showed that LAMP-2A knockdown inhibited the growth of CT26 cells (Fig. 4A) and reduced the Ki67positive cell ratio compared with those in the negative control cells (Fig. 4B and C), confirming decreased cell proliferation following inhibition of CMA. In addition, PCNA, another proproliferation protein, was also down-regulated compared to the level in CTR cells (Fig. 4D and E). These data demonstrated that blocking CMA impeded colon carcinoma cell proliferation. 4. Discussion Autophagy is a catabolic process that functions to degrade
Fig. 1. Immunohistochemical (IHC) staining for LAMP-2A in human tissues from patients with colitis, colorectal adenoma, and different stages of colorectal cancer.
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Fig. 2. Evaluation of chaperone-mediated autophagy (CMA) levels in mouse models of colitis-associated colorectal cancer (CRC). (A) A schematic diagram of model construction. A classical method for generating the CRC model mouse was used, which included intraperitoneal injection of BALB/c mice with a single dose (10.0 mg/kg) of AOM and subsequent administration of 2.5% DSS in drinking water for 7 days (for three cycles). (B) The gross appearance of tumors inside the colon. There were many cancerous tumors in the middle and lower parts of the colon. (C) Immunofluorescence of LAMP-2A (L2A) in CRC (A, white) and tumor-adjacent tissues (B, white). (DeE) Immunoblot for L2A in colon homogenates from CRC and WT mice. *p < 0.05, compared to WT mice, n ¼ 6.
cytoplasmic proteins and organelles in lysosomes, which is crucial for cellular homeostasis and adaptation to various stresses [17,18]. In the past few decades, there has been growing interest in the impact of autophagy on cancer pathogenesis and treatment [19]. Three different autophagic pathways coexist in all cells: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [20]. Previous studies payed much more attention to macroautophagy, which has been shown to function as a tumor suppressor pathway, probably due to its role in the maintenance of genome stability [6,7]. However, the role of macroautophagy in cancer has always been controversial, as it remains unclear whether macroautophagy is good or bad for tumors, and both may be true. Similar confusion arises regarding the impact of macroautophagy in cancer treatment [8]. However, recent studies have made great progress in sorting out this controversy by focusing on CMA, another equally important but less-well-studied form of autophagy [4,8,21e24]. It was shown that there is cross-talk between these autophagic pathways that allows one pathway to compensate for the other [25], thus it may not be sufficient to only study macroautophagy. CMA is an autophagic pathway that is distinct from macroautophagy, and in CMA, only proteins that
contain a specific pentapeptide motif (KFERQ) are degraded [1,2]. Recent studies have implicated CMA in lung cancer [4] and gastric cancer [5]. In addition, CMA activity was shown to be elevated in a variety of cancer cell lines and human cancers of different origins [2e4], independent of the status of macroautophagy [4]. CMA blockade inhibited cancer cell proliferation in vitro [4,5] and led to tumor shrinkage and reduced metastasis in vivo in mice [4]. These data suggest that CMA might play a role in cancer that is unique from that of macroautophagy. Therefore, more research is needed to verify and clarify the role of CMA in cancer. In a previous study of CMA in gastric cancer, the author mentioned CRC, showing that LAMP-2A, the limiting component in CMA, was increased in human CRC [5]. This study prompted us to further investigate the role of CMA in CRC. We analyzed CMA levels in CRC tissue samples of different stages and found that LAMP-2A was almost undetectable in tissues from patients with colitis, but was significantly increased in patients with Stage II and III CRC. Using mouse models of colitis-associated CRC, we observed that LAMP-2A expression was high in CRC tissues, but was almost undetectable in tumor-adjacent tissues. These results demonstrated that CMA was activated in CRC, as has been described for other
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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Fig. 3. Effect of blocking chaperone-mediated autophagy (CMA) on apoptosis of CT26 colon carcinoma cells. (AeD) CT26 cells were transfected with vector-mediated siRNA targeting a LAMP-2A (L2A)-specific exon or a control siRNA (siNC). LAMP-2 (L2), LAMP-2A (L2A), and LAMP-2B (L2B) were detected using western blotting. (EeF) Assessment of DNA fragmentation using TUNEL assay. (GeJ) Detection of relevant apoptosis-related proteins. After transfection, CT26 cells were treated with TNF-a for 8 h to induce apoptosis. All experiments were repeated three times.*p < 0.05, compared to CTR, n ¼ 5.
tumors. This phenomenon is consistent across tumor types, suggesting its research value. To further explore the CMA-related mechanisms in the pathogenesis of CRC, we selectively inhibited CMA by knocking down LAMP-2A in CT26 cells using RNAi. Our results showed that inhibition of CMA enhanced apoptosis and blocked the proliferation of CT26 colon carcinoma cells. These results suggest a reason for the observed high CMA activity in colon cancer. In other words, colon carcinoma cells with higher CMA activity may have lower levels of apoptosis and higher proliferative capacity. Thus, strategies that decrease CMA activity may offer a new therapeutic approach for CRC.
Nevertheless, this study has some limitations. First, the effect of blocking CMA on tumor growth should be verified in vivo studies. This could be achieved by direct injection of a lentivirus encoding a LAMP-2A shRNA into tumor xenografts. This work is ongoing, and will be performed in the future. Second, the mechanisms underlying the role of CMA in tumorigenesis need to be further illuminated. CMA is responsible for the degradation of certain proteins and maintaining protein quality, which may explain why upregulating CMA provides several survival benefits to the cancer cells, including resistance to hypoxia, oxidative stress, and DNA damage [2]. In addition, which proteins are degraded via the CMA pathway remains to be clarified in future studies.
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Fig. 4. Detection of cell proliferation following CMA blockade. (A) After siRNA transfection, the cells proliferation was assayed by MTT at the indicted time-point. (BeC) Cell proliferation was analyzed by staining for ki-67, a standard marker of tumor cell proliferation. (DeE) Western blotting for the pro-proliferation protein PCNA. *p < 0.05, compared to CTR, n ¼ 5.
5. Conclusion
evidence for a potential treatment for CRC by targeting CMA.
In conclusion, our results demonstrated that CMA activity was increased in both human CRC samples and CRC mouse model tissues. Blockade of CMA impeded colon carcinoma cell proliferation, probably by facilitating cell apoptosis. These results provide new
Funding This work was supported by grants from the National Natural Science Foundation of China (81770436). This reserach was also
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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supported by the National Key Project of Chronic NonCommunicable Diseases of China (No. 2016 YFC1300403). Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Authors’ contributions CWQ and LB conceived and designed the study. PJQ performed the experiments. CZH analyzed and interpreted the results. HSM, YJ and PYQ performed the CRC patients’ tissue sample collection and quality control. BC and QL performed analysis and quality control. PJQ wrote the manuscript. All authors read and approved the final manuscript. Declaration of competing interest The authors declare that they have no competing interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.081. References [1] J.F. Dice, Peptide sequences that target cytosolic proteins for lysosomal proteolysis, Trends Biochem. Sci. 15 (1990) 305e309. [2] S. Kaushik, A.M. Cuervo, The coming of age of chaperone-mediated autophagy, Nat. Rev. Mol. Cell Biol. 19 (2018) 365e381. [3] S. Zhang, B. Hu, Y. You, et al., Sorting nexin 10 acts as a tumor suppressor in tumorigenesis and progression of colorectal cancer through regulating chaperone mediated autophagy degradation of p21Cip1/WAF1, Cancer Lett. 419 (2018) 116e127. [4] M. Kon, R. Kiffin, H. Koga, et al., Chaperone-Mediated Autophagy is required for tumor growth, Sci. Transl. Med. 3 (2011), 109ra117. [5] J. Zhou, J. Yang, X. Fan, et al., Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer, Autophagy 12 (2016) 515e528. [6] R. Mathew, C.M. Karp, B. Beaudoin, et al., Autophagy suppresses tumorigenesis through elimination of p62, Cell 137 (2009) 1062e1075.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells Jie-qiong Peng a, b, Shu-mei Han b, Ze-hao Chen e, Jing Yang b, Yan-qing Pei b, Cong Bao d, Lei Qiao c, Wen-qiang Chen c, *, Bo Liu b, ** a
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China Department of Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China c The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, 107 Wenhuaxi Road, 250012, Jinan, China d Department of Pathology, Pingyi County People’s Hospital, Linyi, Shandong, 273300, China e Shandong First Medical University, Taian, Shandong, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2019 Accepted 13 November 2019 Available online xxx
Chaperone-mediated autophagy (CMA) is one of the three types of autophagy. In recent years, CMA has been shown to be associated with the pathogenesis of several types of cancer. However, whether CMA is involved in the pathogenesis of colorectal cancer (CRC) remains unclear. In this study, we investigated CMA activity in tissue specimens from CRC patients and mouse models of colitis-associated CRC (induced by administration of AOM plus DSS). In addition, we down-regulated CMA in CT26 colon carcinoma cells stably transfected with a vector expressing a siRNA targeting LAMP-2A, the limiting component in the CMA pathway, to explore the role of CMA in these cells. Apoptosis was detected using TUNEL assay, and the apoptosis-related proteins were detected using western blotting. Cell proliferation was assessed using MTT assay, Ki-67 labelling and western blotting for PCNA. We found that LAMP-2A expression was significantly increased in CRC patients and mouse models and varied according to the stage of the disease. Inhibition of CMA in CT26 cells facilitated apoptosis, as evidenced by increased TUNEL immunolabeling, increased expression of Bax and Bnip3, and decreased expression of Bcl-2. Cell proliferation assays showed that inhibition of CMA impeded the proliferation of CT26 cells. These data support the hypothesis that CMA is up-regulated in CRC, and inhibition of CMA may be a new therapeutic strategy for CRC patients. © 2019 Elsevier Inc. All rights reserved.
Keywords: Colorectal cancer Chaperone-mediated autophagy Apoptosis Proliferation
1. Introduction Chaperone-mediated autophagy (CMA) is an autophagic pathway in mammalian cells. In contrast to macroautophagy,
* Corresponding author. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, No. 107, Wen Hua Xi Road, Jinan, Shandong, 250012, China. ** Corresponding author. Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, 440 Jiyan road, 250117, Jinan, China. E-mail addresses: [email protected] (W.-q. Chen), 15553115688@163. com (B. Liu).
another form of autophagy, CMA is more specific, and only proteins that contain a specific targeting motif are degraded [1,2]. In recent years, CMA has been associated with the pathogenesis of several types of cancer, and increasing evidence has shown that CMA activity is increased in different cancer cell lines and various cancers of different origins [2e4]. CMA was also shown to be essential for the proliferation of human lung cancer cells [4] and gastric cancer cells [5]. Furthermore, inhibition of CMA in xenografts led to tumor shrinkage and reduced metastasis in mice [4]. Although autophagy has been well studied, most investigations have been focused on macroautophagy, and several studies have highlighted the controversial role of autophagy in cancer [6,7]. Some recent studies have shed light on this debate by examining the involvement of a less-well-studied type of autophagy, CMA [8].
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Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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However, there are few studies on the role of CMA in cancer, and only lung [4] and gastric cancer [5] have been well studied. Therefore, more research is needed. Colorectal cancer (CRC) is a major cancer with a substantial public health burden. It is the third most common diagnosed cancer in males and the second most common in females [9]. Despite recent advances in diagnosis and treatment, the survival rates for advanced stage CRC remain poor. Therefore, it is of great importance to clarify the underlying mechanisms of CRC and identify novel target molecules. Previous studies have shown that the CMA marker LAMP-2A (L2A) is upregulated in human CRC [5], indicating that CMA might play an important role. To test this hypothesis, we first verified CMA levels in both human specimens collected at different stages and CRC mouse models. In addition, we utilized a lentivirus-associated virus-mediated RNA interference (RNAi) technique to knock down the expression of LAMP-2A, the most important component of the CMA pathway, and investigated the effect of CMA dysfunction on cell proliferation and apoptosis using cultured mouse CT26 cells. 2. Materials and methods 2.1. Ethics statement All animal experimental protocols were approved by the Shandong Cancer Hospital and Institute, Shandong First Medical University, and Shandong Academy of Medical Sciences. Tissues from 45 patients with different stages of colorectal cancer, 15 patients with colorectal adenoma, and 15 patients with colitis were collected at Shandong Cancer Hospital, from 2016 to 2018. This study was approved by the Ethics Committee of the Shandong Cancer Hospital and Institute (SDTHEC20171209). All patients provided written informed consent before any experimental procedures were performed. 2.2. Animals Six-week-old male BALB/c mice (n ¼ 25) were obtained from Charles River Laboratories (Beijing, China) and were housed under a 12 h light/dark cycle. Fifteen of these mice were used to generate models of colitis-associated CRC [10,11]. Briefly, mice were administered AOM (10.0 mg/kg body weight; Sigma-Aldrich) as a single intraperitoneal injection and three subsequent cycles of 2.5% (wt/vol) DSS (molecular weight 36,000e50,000; MP Biomedicals) in drinking water for 7 days each. 2.3. Immunohistochemistry and immunofluorescence At 15 weeks, the mice were sacrificed, and the colorectum was harvested and fixed in 4% paraformaldehyde (PFA). These specimens were frozen in OCT and routinely sectioned into 6 mm-thick slabs for histological staining and immunofluorescence. Tissue sections were stained with hematoxylin-eosin (H&E), and frozen cross sections were immunostained using a rabbit polyclonal antibody against LAMP-2A (ab18528; Abcam) and a FITC-labeled secondary goat anti-rabbit IgG antibody (ZSJB-BIO, China). IHC staining was amplified with peroxidase-conjugated streptavidin complexes (ZSJB-BIO), and peroxidase was detected using AEC (ZSJB-BIO) substrate. The signals were visualized under a Manual Positive Fluorescence Microscope (Y-TV55; Nikon, Japan). 2.4. Cell lines and transfection CT26 colon carcinoma cells were obtained from ATCC and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotics at 37 C
in a humidified atmosphere of 5% CO2. LAMP-2A was silenced in CT26 cells with a lentiviral vector-mediated small interfering RNA (siRNA) obtained from Shanghai Genechem Co., Ltd. The sequences targeted by the siRNAs (si1, si2, and si3) were 50 -GACTGCAGTGCAGATGAAG-30 , 50 -CTGCAATCTGATTGATTA-30 , and 50 -TAAACACTGCTTGACCACC-30 , which are located in exon 8a of LAMP-2A and correspond to bases 1198e1216, 1331e1359, and 1678e1700, respectively [12]. Based on our previous experience, we performed two consecutive transfections at a 48 h interval, and the assays were carried out 48 h after the second transfection.
2.5. Western blotting CT26 cells were washed with cold PBS and homogenized on ice in RIPA lysis buffer (Beyotime, China) supplemented with complete protease inhibitor (Beyotime). Supernatants were collected after centrifugation at 12,000g for 30 min. The protein concentration in the supernatant was determined using the BCA Protein Assay Kit (Beijing Solarbio Science & Technology co., Ltd.). Protein samples were boiled with Laemmli sample buffer for 10 min, separated using 12% SDS-PAGE, and transferred to PVDF membranes (0.22 mm; Bio-Rad). After the membranes were blocked with 5% skim milk in TBST for 1 h, they were incubated overnight at 4 C with primary antibodies against LAMP-2A (Invitrogen), LAMP-2B (ab18529; Abcam), LAMP-2 (ab13524; Abcam), Bnip3 (ab109362; Abcam), Bcl-2 (ab182858; Abcam), and Bax (ab32503; Abcam). The protein levels in the western blots were normalized to b-actin (#4970, CST). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with a Luminescent Image Analyzer (Amersham Imager 600; GE, USA). The intensities of the corresponding protein bands were evaluated by densitometry using Image J. Protein expression was assessed relative to b-actin or other indicated protein.
2.6. Cell proliferation Cell proliferation was tested with MTT kit (Sigma) according to the manufacturer’s instruction and Ki-67 labelling. Slides containing CT26 cells were washed with PBS three times and fixed in PFA for 20 min. The cells of the slides were permeabilized by incubation with 0.3% Triton X-100 for 10 min at room temperature and then incubated with 5% goat serum for 30 min to block nonspecific binding. Next, the slides were incubated with an anti-Ki-67 primary antibody (ab15580; Abcam) for 12 h at 4 C, and then with a FITClabeled secondary goat anti-rabbit IgG antibody (ZSJB-BIO, China) on for 1 h. Nuclei were stained with DAPI, and cell fluorescence was analyzed using fluorescence microscopy (DS-Ri2; Nikon, Tokyo, Japan). The proliferation index was obtained by calculating the ratio of Ki-67-positive cells to the total cells in 10 fields of view. The expression of another pro-proliferation protein, PCNA (ab18197; Abcam), was analyzed using western blotting.
2.7. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The results are expressed as the mean ± SD of three independent experiments. Student’s t-test or one-way ANOVA analysis was used to compare the different treatment groups. Statistical significance was set at P < 0.05.
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3. Results 3.1. Chaperone-mediated autophagy activity is increased in human colorectal cancer samples of different stages LAMP-2A is a key protein in CMA, and its level is the most direct indicator of CMA [13,14]. To investigate CMA levels in human CRC samples of different stages, we performed LAMP-2A immunostaining in tissue samples from patients with colitis and stage IeIII CRC who were diagnosed between 2016 and 2018. The results showed that LAMP-2A expression was significantly correlated with tumor staging (Fig. 1). LAMP-2A was almost undetectable in tissues from colitis patients, was slightly increased in tissues from patients with colorectal adenoma and Stage I CRC, but was significantly higher in tissue samples from patients with Stage II and III CRC (Fig. 1). These results were consistent with previous studies and further verified that human CRC tissues show elevated CMA activity. 3.2. Chaperone-mediated autophagy activity is increased in mouse colorectal cancer tissues To explore the variation in CMA in CRC, we evaluated the LAMP2A levels in mouse models of colitis-associated CRC (Fig. 2A and B). Immunofluorescence analysis of mouse colorectal tissues showed that LAMP-2A expression was high in the CRC tissues but was not observed in the tumor-adjacent tissues (Fig. 2C). Western blotting also showed a significant increase in the LAMP-2A levels in the CRC tissues compared to the levels in tumor-adjacent tissues (Fig. 2D and E). In addition, we also investigated the levels of another LAMP2 isoform, LAMP-2B. Analysis showed that the LAMP-2B levels were very low in both the CRC and tumor-adjacent tissues (Fig. S1). These results, along with the data from the human specimens described above, validated the importance of CMA in CRC and demonstrate that CMA is activated in CRC. 3.3. Blocking chaperone-mediated autophagy enhanced apoptosis in CT26 colon carcinoma cells To better illustrate the role of CMA in colon carcinoma cells, we inhibited CMA by knocking down LAMP-2A expression in CT26 cells using a vector-mediated RNA interference (RNAi) technique. As LAMP-2 undergoes alternative splicing of exon 8, leading to the isoforms LAMP-2A, B, and C, and only the LAMP-2A isoform is exclusively involved in CMA [14,15], we choose RNA sequences that specifically target the LAMP-2A exon [12]. All three of the LAMP-
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2A-specific siRNAs (referred to as si1, si2, and si3) downregulated LAMP-2A expression, although si2 and si3 showed the strongest down-regulation (Fig. 3A and B). LAMP-2B was not changed (Fig. 3A and C) and LAMP-2 was reduced in si2 and si3transfected cells (Fig. 3A and D). Therefore, si3 was used in subsequent experiments. Apoptosis can affect the occurrence and development of tumors, and it is of great significance in the treatment of tumors. Therefore, we investigated the role of CMA in apoptosis induced by TNF-a in CT26 cells. TUNEL assays showed that blocking CMA promoted tumor cell apoptosis, as TUNEL immunolabeling was increased in the si3-transfected cells compared to that in siNC-transfected cells (CTR) (Fig. 3E and F). Both pro-apoptotic (Bax and Bnip3) and antiapoptotic (Bcl-2) proteins were detected using western blotting in control siRNA-transfected (CTR) and si3-transfected cells after TNFa treatment. As shown in Fig. 3GeJ, blocking CMA increased the expression levels of Bax and Bnip3 and decreased those of Bcl-2. These data suggested that the inhibition of CMA enhanced the apoptosis of CT26 colon carcinoma cells. As a previous study showed that there was an intersection between two types of autophagy [16], we also checked the protein levels of the specific macroautophagy markers Beclin1, LC3B, and Atg5 to determine if autophagy initiation and progression were activated. None of these proteins were changed following LAMP-2A knockdown (Fig. S2), which confirms that LAMP-2A is not required for macroautophagy. 3.4. Chaperone-mediated autophagy is required for the proliferation of CT26 colon carcinoma cells As we observed that knock down of LAMP-2A in CT26 cells reduced cell proliferation rates compared to those in siNCtransfected cells, we analyzed cell proliferation using the MTT assay and Ki-67 labelling to validate this result and quantify cell proliferation. The MTT results showed that LAMP-2A knockdown inhibited the growth of CT26 cells (Fig. 4A) and reduced the Ki67positive cell ratio compared with those in the negative control cells (Fig. 4B and C), confirming decreased cell proliferation following inhibition of CMA. In addition, PCNA, another proproliferation protein, was also down-regulated compared to the level in CTR cells (Fig. 4D and E). These data demonstrated that blocking CMA impeded colon carcinoma cell proliferation. 4. Discussion Autophagy is a catabolic process that functions to degrade
Fig. 1. Immunohistochemical (IHC) staining for LAMP-2A in human tissues from patients with colitis, colorectal adenoma, and different stages of colorectal cancer.
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Fig. 2. Evaluation of chaperone-mediated autophagy (CMA) levels in mouse models of colitis-associated colorectal cancer (CRC). (A) A schematic diagram of model construction. A classical method for generating the CRC model mouse was used, which included intraperitoneal injection of BALB/c mice with a single dose (10.0 mg/kg) of AOM and subsequent administration of 2.5% DSS in drinking water for 7 days (for three cycles). (B) The gross appearance of tumors inside the colon. There were many cancerous tumors in the middle and lower parts of the colon. (C) Immunofluorescence of LAMP-2A (L2A) in CRC (A, white) and tumor-adjacent tissues (B, white). (DeE) Immunoblot for L2A in colon homogenates from CRC and WT mice. *p < 0.05, compared to WT mice, n ¼ 6.
cytoplasmic proteins and organelles in lysosomes, which is crucial for cellular homeostasis and adaptation to various stresses [17,18]. In the past few decades, there has been growing interest in the impact of autophagy on cancer pathogenesis and treatment [19]. Three different autophagic pathways coexist in all cells: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [20]. Previous studies payed much more attention to macroautophagy, which has been shown to function as a tumor suppressor pathway, probably due to its role in the maintenance of genome stability [6,7]. However, the role of macroautophagy in cancer has always been controversial, as it remains unclear whether macroautophagy is good or bad for tumors, and both may be true. Similar confusion arises regarding the impact of macroautophagy in cancer treatment [8]. However, recent studies have made great progress in sorting out this controversy by focusing on CMA, another equally important but less-well-studied form of autophagy [4,8,21e24]. It was shown that there is cross-talk between these autophagic pathways that allows one pathway to compensate for the other [25], thus it may not be sufficient to only study macroautophagy. CMA is an autophagic pathway that is distinct from macroautophagy, and in CMA, only proteins that
contain a specific pentapeptide motif (KFERQ) are degraded [1,2]. Recent studies have implicated CMA in lung cancer [4] and gastric cancer [5]. In addition, CMA activity was shown to be elevated in a variety of cancer cell lines and human cancers of different origins [2e4], independent of the status of macroautophagy [4]. CMA blockade inhibited cancer cell proliferation in vitro [4,5] and led to tumor shrinkage and reduced metastasis in vivo in mice [4]. These data suggest that CMA might play a role in cancer that is unique from that of macroautophagy. Therefore, more research is needed to verify and clarify the role of CMA in cancer. In a previous study of CMA in gastric cancer, the author mentioned CRC, showing that LAMP-2A, the limiting component in CMA, was increased in human CRC [5]. This study prompted us to further investigate the role of CMA in CRC. We analyzed CMA levels in CRC tissue samples of different stages and found that LAMP-2A was almost undetectable in tissues from patients with colitis, but was significantly increased in patients with Stage II and III CRC. Using mouse models of colitis-associated CRC, we observed that LAMP-2A expression was high in CRC tissues, but was almost undetectable in tumor-adjacent tissues. These results demonstrated that CMA was activated in CRC, as has been described for other
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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Fig. 3. Effect of blocking chaperone-mediated autophagy (CMA) on apoptosis of CT26 colon carcinoma cells. (AeD) CT26 cells were transfected with vector-mediated siRNA targeting a LAMP-2A (L2A)-specific exon or a control siRNA (siNC). LAMP-2 (L2), LAMP-2A (L2A), and LAMP-2B (L2B) were detected using western blotting. (EeF) Assessment of DNA fragmentation using TUNEL assay. (GeJ) Detection of relevant apoptosis-related proteins. After transfection, CT26 cells were treated with TNF-a for 8 h to induce apoptosis. All experiments were repeated three times.*p < 0.05, compared to CTR, n ¼ 5.
tumors. This phenomenon is consistent across tumor types, suggesting its research value. To further explore the CMA-related mechanisms in the pathogenesis of CRC, we selectively inhibited CMA by knocking down LAMP-2A in CT26 cells using RNAi. Our results showed that inhibition of CMA enhanced apoptosis and blocked the proliferation of CT26 colon carcinoma cells. These results suggest a reason for the observed high CMA activity in colon cancer. In other words, colon carcinoma cells with higher CMA activity may have lower levels of apoptosis and higher proliferative capacity. Thus, strategies that decrease CMA activity may offer a new therapeutic approach for CRC.
Nevertheless, this study has some limitations. First, the effect of blocking CMA on tumor growth should be verified in vivo studies. This could be achieved by direct injection of a lentivirus encoding a LAMP-2A shRNA into tumor xenografts. This work is ongoing, and will be performed in the future. Second, the mechanisms underlying the role of CMA in tumorigenesis need to be further illuminated. CMA is responsible for the degradation of certain proteins and maintaining protein quality, which may explain why upregulating CMA provides several survival benefits to the cancer cells, including resistance to hypoxia, oxidative stress, and DNA damage [2]. In addition, which proteins are degraded via the CMA pathway remains to be clarified in future studies.
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Fig. 4. Detection of cell proliferation following CMA blockade. (A) After siRNA transfection, the cells proliferation was assayed by MTT at the indicted time-point. (BeC) Cell proliferation was analyzed by staining for ki-67, a standard marker of tumor cell proliferation. (DeE) Western blotting for the pro-proliferation protein PCNA. *p < 0.05, compared to CTR, n ¼ 5.
5. Conclusion
evidence for a potential treatment for CRC by targeting CMA.
In conclusion, our results demonstrated that CMA activity was increased in both human CRC samples and CRC mouse model tissues. Blockade of CMA impeded colon carcinoma cell proliferation, probably by facilitating cell apoptosis. These results provide new
Funding This work was supported by grants from the National Natural Science Foundation of China (81770436). This reserach was also
Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081
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supported by the National Key Project of Chronic NonCommunicable Diseases of China (No. 2016 YFC1300403). Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Authors’ contributions CWQ and LB conceived and designed the study. PJQ performed the experiments. CZH analyzed and interpreted the results. HSM, YJ and PYQ performed the CRC patients’ tissue sample collection and quality control. BC and QL performed analysis and quality control. PJQ wrote the manuscript. All authors read and approved the final manuscript. Declaration of competing interest The authors declare that they have no competing interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.081. References [1] J.F. Dice, Peptide sequences that target cytosolic proteins for lysosomal proteolysis, Trends Biochem. Sci. 15 (1990) 305e309. [2] S. Kaushik, A.M. Cuervo, The coming of age of chaperone-mediated autophagy, Nat. Rev. Mol. Cell Biol. 19 (2018) 365e381. [3] S. Zhang, B. Hu, Y. You, et al., Sorting nexin 10 acts as a tumor suppressor in tumorigenesis and progression of colorectal cancer through regulating chaperone mediated autophagy degradation of p21Cip1/WAF1, Cancer Lett. 419 (2018) 116e127. [4] M. Kon, R. Kiffin, H. Koga, et al., Chaperone-Mediated Autophagy is required for tumor growth, Sci. Transl. Med. 3 (2011), 109ra117. [5] J. Zhou, J. Yang, X. Fan, et al., Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer, Autophagy 12 (2016) 515e528. [6] R. Mathew, C.M. Karp, B. Beaudoin, et al., Autophagy suppresses tumorigenesis through elimination of p62, Cell 137 (2009) 1062e1075.
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Please cite this article as: J.-q. Peng et al., Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.081