Fibronectin Type III Domain Containing 5 Contributes to Nab-paclitaxel Chemoresistance by Promoting Autophagy via the AMPK/mTOR Signaling Pathway in Hepatocellular Carcinoma Cells

doi:10.21203/rs.3.rs-2130095/v1

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Fibronectin Type III Domain Containing 5 Contributes to Nab-paclitaxel Chemoresistance by Promoting Autophagy via the AMPK/mTOR Signaling Pathway in Hepatocellular Carcinoma Cells

https://doi.org/10.21203/rs.3.rs-2130095/v1

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Chemotherapy resistance is a huge challenge in the treatment of hepatocellular carcinoma because resistance to nab-paclitaxel largely affects the efficacy of chemotherapy. An increased expression of fibronectin type III domain containing 5 (FNDC5) in hepatocellular carcinoma cells can predict post-hepatectomy complications in patients with hepatocellular carcinoma and also stimulate proliferation and invasion of hepatocellular carcinoma cells; however, its role in the chemotherapy of hepatocellular carcinoma cells has never been evaluated. Thus, this study aimed to explore whether FNDC5 regulates chemoresistance in hepatocellular carcinoma. We identified by immunohistochemistry that hepatocellular carcinoma tissues had a higher FNDC5 expression than normal tissues adjacent to the cancer cells. Subsequently, knockdown of FNDC5 in hepatocellular carcinoma cells resulted in their diminished resistance to cell death after chemotherapy with nab-paclitaxel. By contrast, overexpression of FNDC5 in hepatocellular carcinoma cells increased the resistance of hepatocellular carcinoma cells to treatment. Moreover, FNDC5 mechanistically promoted autophagy via the AMPK/mTOR signaling pathway, thereby reducing cell death induced by nab-paclitaxel. Finally, we tested our hypothesis by conducting animal experiments. In conclusion, FNDC5 could be used as a biomarker for predicting chemotherapeutic efficacy in hepatocellular carcinoma treated with nab-paclitaxel chemotherapy, and as a therapeutic target to overcome resistance to nab-paclitaxel in hepatocellular carcinoma chemotherapy.

fibronectin type III domain containing 5

autophagy

hepatocellular carcinoma

AMPK/mTOR pathway

albumin-bound paclitaxel

Hepatocellular carcinoma (HCC) is the world's sixth most prevalent malignancy and the third most common cause of cancer-related mortality. HCC is the most common primary liver tumor, and its prognosis remains poor compared to other tumor entities (1). Generally, most patients with HCC are diagnosed at an incurable tumor stage and lost to surgery (2–4). In addition to the common surgical methods and liver transplantation, liver cancer can also be treated with chemotherapy, which is one of the most common therapies for liver cancer, especially in advanced stages (5).

Albumin-bound paclitaxel (nab-paclitaxel) is an effective chemotherapeutic agent that has been successfully used extensively in the chemotherapy of various tumors, including endometrial, ovarian, and lung cancers, due to its unique anti-cancer mechanism. Similarly, it is also indicated for the chemotherapy of HCC (6–9). Although nab-Paclitaxel is not a first-line drug for the treatment of HCC, several studies have shown that it has a very promising future for the treatment of HCC. It is an antimitotic medication that encourages tubulin polymerization and prevents mitosis from progressing (10, 11).

However, most patients with HCC often face ineffective or progressively ineffective chemotherapy due to chemotherapy resistance in HCC cells, which remains one of the major barriers to effective HCC treatment (12, 13), Multiple mechanisms contribute to the complex phenomenon of drug resistance (14). These mechanisms allow HCC cells to continue to proliferate even in chemotherapy treatment with nab-paclitaxel, which may lead to failure of chemotherapy treatment. Therefore, investigating the mechanisms of resistance to nab-paclitaxel in HCC is particularly important. However, studies on the resistance against nab-paclitaxel in HCC and biomarkers that can predict the response to nab-paclitaxel chemotherapy in patients with HCC are scarce, and the need to explore such a biomarker is urgent.

We hypothesized that fibronectin type III domain containing 5 (FNDC5) is one such biomarker. Traditionally, FNDC5 has been shown to be present in the muscle as a myokine that drives the browning of white fat and increases energy expenditure and heat dissipation, so it is considered in the treatment for human metabolic disorders (15, 16). FNDC5 is processed by protein hydrolysis to release into the circulation irisin, a cytokine isolated from muscle by Bostrom et al. (17). FNDC5 has since been reported to have roles not only in different tissues and organs, in addition to muscle tissues, but also in the adipose tissues, bones, and nerves (18). FNDC5 is also present in various pathological processes, such as cancer processes and inflammatory processes (19, 20). FNDC5 has also recently been reported to improve insulin resistance and is involved in several cardiovascular diseases such as cardiac hypertrophy and atherosclerosis (21–23). It plays an equally crucial role in various oncological diseases. We previously discovered that FNDC5 is substantially expressed in HCC tissues, and it encourages the malignant proliferation of HCC cells (24). However, studies using FNDC5 as a biomarker in HCC cells to study the chemoresistance of HCC and to predict the response to chemotherapy patients with HCC are few.

Autophagy is the process by means of which cells use lysosomes to destroy their own organelles and macromolecules under the control of autophagy-related genes. It has an important role in achieving the metabolic needs of cells, renewing certain organelles, and maintaining intracellular homeostasis. Autophagy is classified as macroautophagy, microautophagy, and chaperone-mediated autophagy. Of these, macroautophagy is the most dominant of the three forms and consists of lipid membrane structures that wrap around the material to be degraded to form autophagosomes. Subsequently, the autophagosome merges with the lysosome and after going through this process the contents of it are degraded (25–28). Autophagy is reportedly associated with tumorigenesis and chemoresistance, thus making it an important mechanism leading to chemoresistance (29). In autophagy, AMPK is not only an essential kinase for modulating energy homeostasis, but also one of the critical proteins participating in several signalling pathways (30). However, the mechanism by which FNDC5 affects HCC chemoresistance through autophagy remains unclear. Therefore, this study aimed to investigate how FNDC5 regulates chemoresistance to nab-paclitaxel in HCC.

2.1 Ethics approval

Patient tumor tissue specimen collection was performed in full compliance with the provisions of the research ethics committee of Qingdao Municipal Hospital. Furthermore, all immunohistochemistry experiments were conducted under the supervision of the research ethics committee staff. The experimental protocol for the animals was designed rigorously in compliance with the ARRIVE guidelines and validated by the research ethics committee of the abovementioned hospital. All experimental procedures were recorded, reviewed, and completed under the supervision and guidance of the said ethics committee (ethics approval number: 104).

2.2 Reagents and antibodies

The anti-FNDC5 antibody (Cat.ab181884) was purchased from Abcam (Cambridge, UK). The anti-LC3B antibody (Cat.L7543) was purchased from Sigma-Aldrich (MO, USA). Antibodies against P62 (Cat.#5114), Beclin-1 (Cat.#3495), mTOR (Cat.#2983), phospho-mTOR (Cat.#2974), AMPKα (Cat.#5831), phospho-AMPKα (Cat.#2535), and β-actin (Cat.#4967) were purchased from Cell Signaling Technology (MA, USA). Secondary antibodies-goat anti-rabbit mouse IgG-horseradish peroxidase (HRP) (Cat.M21003) was purchased from Abmart (Shanghai, China). Dorsomorphin (Cat.HY-13418A) was purchased from MCE (NJ, USA).

2.3 Cell lines and culture conditions

The Chinese Academy of Sciences' Cell Bank (Shanghai, China) supplied the human HCC cell lines HepG-2 and SMMC7721. The above cells were cultured in Dulbecco's modified Eagle's medium which contained 10% fetal bovine serum (FBS, Excellbio, USA) and 1% penicillin–streptomycin (HyClone, UT, USA). All the cells were incubated at 37°C with 5% CO₂.

2.4 FNDC5 knockdown and overexpression

Human FNDC5 cDNA was prepared with Gene Chem (Shanghai, China), which was packaged in a lentiviral vector, and used for cell transfection. The overexpressed FNDC5 and negative controls were named FNDC5–OE and FNDC5–NC1, respectively. FNDC5 was knocked down using siRNA produced by RIBOBIO. GGAGGATACGGAGTACATA, AGAAGATGGCCTCCAAGAA, and GCTTCATCCAGGAGGTGAA were the siRNA sequences. The first and second siRNAs with more effective knockdown were selected, and plasmids with hU6-MCS-CBhgcGFP-IRES-puromycin component sequences were used, packaged as lentiviral vectors, and these were done by Gene Chem. FNDC5–KD1, FNDC5–KD2, and FNDC5–NC2 were the knockdown and negative controls, respectively. After transfection of HepG-2 and SMMC7721 cell lines according to the prescribed steps, the successfully transfected cells were screened with 1 µg/mL of puromycin for use in further experiments.

2.5 Cell treatments

Nab-paclitaxel (Abraxane, Abraxis BioScience, Los Angeles, CA, USA) was dissolved in sterile 0.9% NaCl and administered to the cell culture as a concentrate (1 mg/mL). The control wells received the same amount of solvent (sterile 0.9% NaCl). The cells were harvested at the conclusion of each time point and used in the following studies. For these tests, the floating cells were collected by centrifugation and removal of the supernatant, then mixed with retrieved adherent cells.

In the AMPK inhibition trials, specific inhibitors for individual AMPK (Dorsomorphin, MCE) were utilized. Prior to the nab-paclitaxel therapy, a 1-hour pre-treatment with Dorsomorphin was performed. Instead of the AMPK inhibitors, dimethylsulfoxide (DMSO) was used as the control solvent in each control well.

2.6 Immunohistochemistry

Immunohistochemistry methods were used as our team did in previous experiments (31, 32). Tumor samples from 60 individuals with liver cancer were obtained and fixed in 4.0% paraformaldehyde before being embedded in paraffin. Next, the tissue wax blocks were sliced into 4-mm slices and treated with 3.0% hydrogen peroxide before being sealed with 5.0% FBS. They were incubated overnight at 4°C with the FNDC5 antibody (1:200, ab181884, Abcam, Cambridge, MA, USA) and AMPK antibody (1:250, ab32047, Abcam, Cambridge, MA, USA), followed by secondary antibodies coupled to species-specific HRP and detection with diaminobenzidine with hematoxylin as a reverse stain. The AxioVision Rel.4.6 computerized image analysis system was used for the final photographs (Carl Zeiss). The degree of tissue staining (area × intensity) was assessed, with the staining area scored as follows: 0 (staining area close to 0%), 1 (staining area < 10%), 2 (staining area 10–35%), 3 (staining area 35–75%), and 4 (staining area 76–100%). The following were the staining intensity scores: 0 indicates no staining, 1 indicates faint staining, 3 indicates moderate staining, and 4 indicates severe staining (strong staining). The final degree of protein expression was compared using SI scores (SI = staining area score × staining intensity score), with SI ≥ 8 indicating a high expression and SI < 8 indicating a low expression.

2.7 Confocal immunofluorescence

The cells were cultivated on cover slips and permeabilized with 0.1% Triton X-100 after being fixed with 4% paraformaldehyde for 15 min. Primary anti-LC3B antibodies (1:100, Sigma-Aldrich, MO, USA) diluted in 1% bovine serum albumin (BSA) were applied overnight at 4°C after blocking in 3% BSA for 30 min. After three polybutylene succinate washes, the slides were incubated for 1.5 h at room temperature with an Alexa Fluor 488-conjugated secondary antibody (1:1000, Invitrogen). LC3B puncta pictures were obtained using an inverted confocal fluorescence microscope after the cells were counterstained with DAPI (Carl Zeiss, Germany).

2.8 Flow cytometry

A FITC Annexin V Apoptosis Detection Kit (BD Biosciences, CA, USA) was used to analyze cell apoptosis in HCC cells. In accordance with the guidelines, the cells were analyzed on a FACScan flow cytometer (Beckman Coulter Inc., CA, USA) using CellQuest and the FlowJo software.

2.9 Western blotting

A western blotting analysis was performed by our team as in the previous experiments (31, 32). The total proteins were recovered from HCC cells by lysis with radioimmunoprecipitation assay buffer in a 4°C environment. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) was used to separate the proteins, which were then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight. As an internal reference, the membranes were treated with β-actin. Diluted HRP conjugated secondary antibodies were then added and incubated for 2 h at room temperature. Finally, protein blots were detected using enhanced chemiluminescence detection reagents (Millipore, MA, USA). The ImageJ software was used to examine the intensities of the protein blots, which were gathered from three separate studies.

2.10 Xenograft mouse model

As with our team's previous approach (31, 32), the Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) provided the NOD-SCID (NOD CB17-Prkdcscid/NcrCrl, male, 5 weeks old) mice. The mice were housed in a 12 h light/dark cycle environment set at 25 ± 1 ℃ and 56% humidity and were provided free access to food and water. The trials were conducted on twenty mice weighing between 20 and 23 g at baseline. The mice were intially subcutaneously injected in their backs with 5 × 10⁶ SMMC-7721 cells, and then divided into the following four groups: control (n = 5), FNDC5 overexpression (n = 5), FNDC5 overexpression followed by treatment with AMPK inhibitor Dorsomorphin (1 mg/kg, MCE, China) (n = 5), and FNDC5 knockdown groups (n = 5). Lyophilized nab-paclitaxel (Abraxane, Abraxis BioScience, Los Angeles, CA) was reconstituted in sterile endotoxin-free saline to a concentration of 2.5 mg/ml. The mice received 0.1 ml, which corresponds to a 10 mg/kg dose for a 25-g mouse. The nab-paclitaxel treatment started only when the tumor size was 0.125 cm³. The mice were injected intraperitoneally with nab-paclitaxel once every 3 days over 21 days. DMSO was used to dilute the Dorsomorphin. Dorsomorphin was delivered intraperitoneally every other day to the mice in the third group at 7 days following nab-paclitaxel injection, whereas the other groups received regular saline injections. The tumor volume was measured every 3 days,. The mice were euthanized by cervical dislocation on day 21, and the tumors were removed.

2.11 Statistical analysis

For data analysis, the GraphPad Prism 9 software (GraphPad Software Inc., CA, USA) was utilized. All experimental results are presented as the mean ± SEM and are based on data from at least three separate duplicate trials. The t-test was used to compare differences between two groups. A one-way analysis of variance was used to compare data from different experimental groups. Significance was set at P-value < 0.05.

3.1 Correlation of FNDC5 level in HCC cells with chemoresistance to nab-paclitaxel

In examining FNDC5 expression by immunohistochemistry, FNDC5 had a higher expression in HCC tissues than in paracancerous tissues (Fig. 1A).

The expression of FNDC5 protein was increased after using sorafenib and nab-Paclitaxel to act on HepG-2 cells, respectively. This suggests that sorafenib and nab-Paclitaxel may be associated with FNDC5. Our group has confirmed the relationship between sorafenib and FNDC5 in other studies, and the present study aimed to investigate the relationship between nab-Paclitaxel and FNDC5 (Fig. 1B) (32).

After treating HepG-2 and SMMC-7721 cells with nab-paclitaxel, the expression of FNDC5 protein was increased after 24 and 48 h of nab-paclitaxel action on HepG-2 and SMMC-7721 cells. Thus, FNDC5 may be implicated in the nab-paclitaxel response (Fig. 1C).

The percentage of apoptotic cells increased with additional doses of nab-paclitaxel in HepG-2 and SMMC-7721 cells after treatment with varied concentrations of nab-paclitaxel for 24 h, according to Annexin-V/PI staining and flow cytometry. In the subsequent experiments, the lowest nab-paclitaxel concentration (10 µg/ml) that caused significant apoptosis in HepG-2 and SMMC-7721 was used (Fig. 1D and E).

3.2 Knockdown of FNDC5 in HCC increases the drug sensitivity of nab-paclitaxel

In two cell lines, HepG-2 and SMMC-7721, we knocked down the FNDC5 gene. The reduced expression of FNDC5 protein levels indicates the successful generation of FNDC5 knockdown cells (Fig. 1F). To evaluate the regulatory character of FNDC5 in regulating nab-paclitaxel-mediated responses in HepG-2 and SMMC-7721 cells, the HCC cells in the vector group or FNDC5 knockdown group were treated with 10 µg/ml of nab-paclitaxel to detect the percentage of apoptotic cells. After 24 h of nab-paclitaxel treatment, approximately 5% of the cells in the vector group underwent apoptosis. By contrast, the apoptosis rate in the FNDC5 knockdown group was approximately 15%, which was considerably greater than that of the cells in the vector group (Fig. 1G). Thus, FNDC5 levels in HCC cells may be positively correlated with resistance to nab-paclitaxel.

3.3 FNDC5 induces autophagy in HCC cells

The level of LC3 protein expression was positively linked with the level of autophagy (33). We observed that autophagy occurred in nab-paclitaxel-treated HCC cells by examining changes in the western blot analysis of autophagy-related markers before and after FNDC5 knockdown at different times of nab-paclitaxel treatment. The LC3 and Beclin1 expression levels increased, while the P62 expression levels decreased. Additionally, using immunofluorescence to detect LC3 expression in HCC cells confirmed the abovementioned findings. We then knocked out FNDC5, reversing this abovementioned phenomenon. These were sufficient to demonstrate that FNDC5 increased the level of autophagy, leading to nab-paclitaxel-induced drug resistance (Fig. 2A–C).

3.4 FNDC5-induced autophagy is mediated by the AMPK/mTOR signaling pathway

One of the key mechanisms of autophagy activation is AMPK activation and mTOR inhibition (34, 35). We inspected the activation of AMPK/mTOR pathway before and after FNDC5 knockdown by western blot at different times of nab-paclitaxel treatment. Phosphorylation levels of AMPK increased, while those of mTOR expectedly decreased, although the expression levels of overall AMPK and mTOR were unaffected. Moreover, subsequent knockdown of FNDC5 attenuated this activation (Fig. 3A and B).

Regarding the relationship between FNDC5 and AMPK in HCC tissues, the FNDC5 protein levels were positively correlated with AMPK levels in HCC patients (Fig. 3C).

3.5 Overexpression of FNDC5 renders nab-paclitaxel resistant in HCC cells via the AMPK pathway

Our experiments demonstrated the overexpression of the FNDC5 gene in HepG-2 and SMMC-7721. The increased expression of FNDC5 protein levels indicates a successful overexpression (Fig. 4A). The apoptotic rate of HCC cells in the overexpression group was lower than that of HCC cells in the vector group (Fig. 4B). Subsequently, we examined the changes in the levels of key marker proteins in autophagy and in the AMPK/mTOR pathway before and after FNDC5 overexpression after 24 h of nab-paclitaxel treatment. We observed that overexpression of FNDC5 enhanced nab-paclitaxel-induced autophagy levels and nab-paclitaxel-induced activation of AMPK and mTOR (Fig. 4C and D).Regarding the inhibition of AMPK phosphorylation before and after FNDC5 overexpression using the AMPK inhibitor Dorsomorphin (Fig. 4E), overexpression of FNDC5 in cell lines without the AMPK inhibitor Dorsomorphin reduced the drug sensitivity of HCC cells to nab-paclitaxel, while in cell lines with the AMPK inhibitor Dorsomorphin, overexpression of FNDC5 did not reduce its drug sensitivity, demonstrating that FNDC5 resistance to nab-paclitaxel is AMPK-dependent (Fig. 4F).

3.6 In vivo experimental study of the effect of FNDC5 expression level on nab-paclitaxel chemotherapy resistance in HCC

Regarding the chemoresistance of nab-paclitaxel in the treatment of HCC, tumors in the FNDC5 knockout group grew slowly, while those in the high FNDC5 expression group grew rapidly. Tumors in the FNDC5 overexpression group, which was also injected with the AMPK inhibitor Dorsomorphin, were smaller than those in the FNDC5 overexpression group (Fig. 5A and B). Thereafter, we crushed the tumors and extracted the proteins, which were then used to examine the protein expression related to autophagy by western blot. The findings indicated that autophagy levels were reduced in the FNDC5 knockout group, while those in the high FNDC5 expression group increased. The FNDC5 overexpression group, which was also injected with the AMPK inhibitor Dorsomorphin, had lower autophagy levels than the FNDC5 overexpression group (Fig. 5C). These results further suggest that FNDC5 promotes autophagy in HCC cells through the AMPK pathway and leads to chemoresistance of albumin paclitaxel in treating HCC (Fig. 5D).

Chemotherapeutic agents such as nab-paclitaxel and sorafenib remain a key drug for treating many solid tumors, including HCC. However, the development of chemoresistance is among the main causes for therapy failure. Our team has explored resistance to sorafenib in the treatment of HCC in other studies, and the aim of this study is to investigate the causes of resistance to nab-Paclitaxel in HCC (32). Understanding the main mechanisms underlying the development of chemoresistance to nab-paclitaxel in HCC and exploring biomarkers that measure or influence this chemotherapy resistance are essential to reverse this failure-causing situation and to develop new effective treatments. The AMPK/mTOR pathway has been reported to play an important role in the response of HCC to some chemotherapeutic agents (36). The chemotherapeutic effect of tumors is altered in the presence of altered expression of FNDC5 (37). Because chemoresistance to nab-paclitaxel is a significant barrier to HCC treatment, this study’s findings may be helpful in uncovering the mechanisms of chemoresistance to nab-paclitaxel in treating HCC. This has never been confirmed by a study before our research. Moreover, the important role played by FNDC5 in the response to nab-paclitaxel chemotherapy in HCC in this study demonstrates that FNDC5 is a fully feasible marker for predicting the effects of nab-paclitaxel chemotherapy in HCC. Hence, FNDC5 could also naturally be an important target worth developing to improve the efficacy of chemotherapy in HCC.

In the histochemistry, FNDC5 was differentially expressed in HCC cells and normal paraneoplastic tissues, with HCC cells express FNDC5 at a higher level than normal paraneoplastic tissues. Our study showed enhanced expression of FNDC5 protein after the use of sorafenib and nab-Paclitaxel in HepG-2 cells, respectively, suggesting that the enhanced expression of FNDC5 is associated with the above two chemotherapeutic agents. In this study, nab-Paclitaxel was selected as our study subject for HCC chemoresistance. Additionally, we observed that the FNDC5 expression changed in SMMC7721 and HepG2 by different times of nab-paclitaxel treatment, and the longer the time of nab-paclitaxel treatment of SMMC7721 and HepG2, the higher the degree of FNDC5 expression. These findings imply that FNDC5 is implicated in the nab-paclitaxel response. To further elucidate the function of FNDC5 in response to chemotherapy in HCC, we knocked down FNDC5 in SMMC7721 and HepG2 with a lentivirus. Similarly, we overexpressed FNDC5 in SMMC7721 and HepG2 using a lentiviral method. Knockdown of FNDC5 in SMMC7721 and HepG2 increased its chemotherapeutic drug sensitivity to nab-paclitaxel, whereas the overexpression of FNDC5 in SMMC7721 and HepG2 reduced its chemotherapeutic drug sensitivity to nab-paclitaxel. This was also observed in a xenograft mouse model for in vivo animal experiments (Fig. 5A–C). This suggests that the expression level of FNDC5 may be positively correlated with the chemoresistance of nab-paclitaxel in HCC cells and may indicate the sensitivity of HCC to nab-paclitaxel chemotherapy. Therefore, FNDC5 may have a role in enhancing the chemoresistance of HCC to nab-paclitaxel.

In HCC cells with a high expression of FNDC5, enhanced resistance to nab-paclitaxel was associated with the activation of autophagic mechanisms, suggesting that autophagy in HCC cells inhibited cell death after nab-paclitaxel chemotherapy. We confirmed a direct link between FNDC5 and autophagy. Furthermore, knockdown of FNDC5 in HCC cells resulted in diminished autophagy, which was associated with apoptosis due to nab-paclitaxel chemotherapy. Other studies have reported that nab-paclitaxel can affect the level of autophagy in various kinds of cancer cells and that the occurrence of these autophagy has a significant impact in determining the drug sensitivity of cancer cells to nab-paclitaxel (38–40). Apoptosis and autophagy can promote, antagonize, or assist each other, thus affecting the final fate of the cell (41). For example, other studies have revealed that vitamin K2 can induce both autophagy and apoptosis in leukemic cells (42). Other studies have also reported that autophagy can be induced to promote apoptosis in cervical cancer cells (43). Here, we confirmed that autophagy exists as a mechanism to promote chemotherapy resistance of nab-paclitaxel in HCC cells, and using FNDC5 to regulate the level of autophagy helps to control the apoptosis level in HCC cells. Moreover, autophagy-mediated chemoresistance occurred in HCC cells after nab-paclitaxel treatment, which was reversed after knockdown of FNDC5. Thus, our findings suggest an extremely strong causal relationship between apoptosis and autophagy, and in our study, FNDC5 enhanced the level of autophagy in HCC cells and reduced the apoptosis induced by nab-paclitaxel chemotherapy, leading to chemoresistance of nab-paclitaxel in the treatment of HCC.

As an important kinase regulating energy homeostasis, AMPK is one of the central regulators of metabolism in eukaryotic cells and organisms, maintaining the smooth function of cellular physiological activities, and it is also a key protein involved in various signaling pathways (44, 45). AMPK/mTOR, a key pathway involved in multiple signaling, is an important central regulator of autophagy (46). AMPK is activated and may trigger autophagy by phosphorylating and inactivating mTOR (47). Furthermore, our research experiments revealed that overexpression of FNDC5 in cell lines without the AMPK inhibitor Dorsomorphin reduced the drug sensitivity of HCC cells to nab-paclitaxel. However, in cell lines using the AMPK inhibitor Dorsomorphin, overexpression of FNDC5 did not reduce their drug sensitivity, and these results provide further evidence that FNDC5 causes resistance to nab-paclitaxel in HCC treatment through the AMPK/mTOR pathway.

There are still some limitations in this research. Nab-paclitaxel is not a first-line drug for HCC chemotherapy, so we have great difficulty in collecting a large sample of such patients. Therefore, many of the current studies do not rise to a higher level, and we can only prove our point through other basic studies. We will try to collect such patients as much as possible in our follow-up studies to supplement the limitations of our study.

Nevertheless, this research revealed that FNDC5 promotes autophagy in HCC cells through the AMPK/mTOR pathway and leads to chemoresistance of nab-paclitaxel in HCC treatment and affects apoptosis of HCC cells induced by nab-paclitaxel chemotherapy. To the best of our knowledge, this study is the first to confirm the significant role of FNDC5 in HCC leading to nab-paclitaxel chemoresistance through autophagy. Thus, FNDC5 could serve as a marker to predict the efficacy of nab-paclitaxel chemotherapy in HCC and could also be developed as an important target for therapeutic approaches to improve the efficacy of nab-paclitaxel chemotherapy in HCC.

BSA, bovine serum albumin

FBS, fetal bovine serum

FNDC5, fibronectin type III domain containing 5

HCC, hepatocellular carcinoma

HRP, horseradish peroxidase

DMSO, dimethylsulfoxide

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author contributions

Y.Z.: conceptualization, investigation, formal analysis, writing-original draft. J.P.: methodology, resources. H.L.: formal analysis, writing-review and editing. W.C.: resources. J.C.: supervision, funding acquisition. G.S.: supervision, conceptualization ,project administration, writing review and editing, funding acquisition. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by research grants from the Shandong Province Key Research and Development Project (NO. 2018G SF118057); Chen Xiao-ping Foundation for the Development of Science and Technology of Hubei Province (CXPJJH11900001-2019206).

Acknowledgments

We would like to thank Editage (www.editage.cn) for English language editing.

Contribution to the field statement

This study demonstrates that fibronectin type III domain containing 5 plays an essential role in nab-paclitaxel chemotherapy resistance in hepatocellular carcinoma. This makes fibronectin type III domain containing 5 promising in predicting the efficacy of nab-paclitaxel, and in the future, it could also be developed as a target to improve the therapeutic efficacy of nab-paclitaxel.

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31 Oct, 2022
Reviewers invited by journal
30 Oct, 2022
Editor assigned by journal
06 Oct, 2022
First submitted to journal
03 Oct, 2022

You are reading this latest preprint version

Fibronectin Type III Domain Containing 5 Contributes to Nab-paclitaxel Chemoresistance by Promoting Autophagy via the AMPK/mTOR Signaling Pathway in Hepatocellular Carcinoma Cells

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Ethics approval

2.2 Reagents and antibodies

2.3 Cell lines and culture conditions

2.4 FNDC5 knockdown and overexpression

2.5 Cell treatments

2.6 Immunohistochemistry

2.7 Confocal immunofluorescence

2.8 Flow cytometry

2.9 Western blotting

2.10 Xenograft mouse model

2.11 Statistical analysis

3. Results

3.1 Correlation of FNDC5 level in HCC cells with chemoresistance to nab-paclitaxel

3.2 Knockdown of FNDC5 in HCC increases the drug sensitivity of nab-paclitaxel

3.3 FNDC5 induces autophagy in HCC cells

3.4 FNDC5-induced autophagy is mediated by the AMPK/mTOR signaling pathway

3.5 Overexpression of FNDC5 renders nab-paclitaxel resistant in HCC cells via the AMPK pathway

3.6 In vivo experimental study of the effect of FNDC5 expression level on nab-paclitaxel chemotherapy resistance in HCC

4. Discussion

5. Nomenclature

Declarations

References

Status:

Version 1