Plasmodium infection prevents recurrence and metastasis of hepatocellular carcinoma potentially via inhibiting EMT

Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer mortality worldwide and is characterized by a high rate of recurrence. We have previously reported Plasmodium infection inhibits tumor development and metastasis in a murine Lewis lung cancer model. In the current study, we aimed to examine the effects of Plasmodium infection on HCC metastasis and recurrence. Methods: Antitumor effects of Plasmodium infection were determined using two murine orthotopic HCC models, the non-resection model for investigating the effect of Plasmodium infection on liver tumor progression and metastasis, the resection model for investigating the effect of Plasmodium infection on the tumor recurrence after tumor was removed. Tumor tissues derived from tumor-bearing mice treated with or without Plasmodium infection were harvested after 15 days of tumor inoculation. The biomarkers related to epithelial-mesenchymal transition (EMT) and molecules associated with CCR10-mediated PI3K/Akt/GSK-3β/Snail pathway signaling were identied by qRT-PCR and western blot. Results: We found that Plasmodium infection signicantly suppressed progression, recurrence and metastasis of HCC and prolonged the survival of tumor-bearing mice in both models. The expression levels of E-cadherin were signicantly higher in Plasmodium treated group compared with those in control group, whereas the expression levels of Snail were signicantly lower in the treated group than those in control group. Furthermore, Plasmodium infection inhibited the activation of Akt and GSK-3β in the tumor tissues by downregulating the expression of CCR10, thereby suppressing the accumulation of Snail and potentially contributed to the suppression of EMT and the prevention of tumor recurrence and metastasis. Conclusion: This study suggested that Plasmodium infection inhibited recurrence and metastasis, improved the prognosis of HCC potentially via suppression of CCR10 ‐ mediated PI3K/Akt/GSK ‐ 3β/Snail signaling, and prevention of EMT. These ndings may be important in the development of novel therapy for HCC recurrence and metastasis especially for patients during the perioperative period.


Introduction
Hepatocellular carcinoma (HCC) ranks fourth in the cause of cancer-related death worldwide. The annual incidence of this disease is believed to be on the rise, especially in Asia [1]. Surgical resection is still one of the main therapies for HCC, and the characteristics of early postoperative recurrence are indicative for the prognosis of this disease [2]. The HCC recurrence rate of 2 years after the operation is as high as 61.6% which is the leading cause of cancer-associated death according to the statistics of the World Health Organization [3]. Many factors correlate to the distant recurrence of liver cancer, for instance, serum AFP over 400ng/ml and tumor size larger than 5cm. Microvascular and portal vein cancer thrombus indicate distant metastasis of tumor cells that pass through vascular basement membrane after metamorphosis, and may contribute to HCC postoperative intrahepatic invasion in the future [4]. At present, the metastasis of liver cancer cells cannot be completely blocked by surgical resection and effective drugs are lacking.
Epithelial-mesenchymal transition (EMT) is the process that epithelial cancer cells lose their polarity and change into motile mesenchymal cells, which is an important mechanism leading to distant metastasis of malignant tumors. EMT induces cancers metastasis through promoting the separation and invasion of cancer cells from homologous cells [5]. It has been demonstrated that E-cadherin can maintain the tight connection between cells and prevent cell invasion and metastasis, and the decrease of E-cadherin is a common EMT biomarker [6]. The upregulation of transcription factor Snail can also be used as a common biomarker of EMT [7,8].
Evidence suggests that chemokines/chemokine receptors play key roles in the invasion and metastasis of malignant tumors [9][10][11]. Chemokines act as prominent recruiters in the invasive process of cancer cells. Through the effects of chemokines, tumor advantageous factors such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) accumulate around the tumor cells, therefore altering the microenvironment of tumors. One type of GPCR, CC-chemokine receptors 10 (CCR10), has been found to be signi cantly up-regulated in HCC tumors. CCR10 activates the phosphorylation of downstream signal molecules to promote EMT. Furthermore, PI3K/Akt/GSK-3β has been demonstrated to be a classical signaling pathway for CCR10 to activate intracellular signaling molecules [11].
Our previous studies have demonstrated that Plasmodium infection signi cantly inhibits tumor growth and prolongs the survival time of lung cancer-bearing mice through activating the innate and adaptive anticancer immunity, remodeling the tumor immunosuppressive microenvironment, such as reducing the numbers of MDSCs and Tregs within tumor tissue, and inhibiting tumor angiogenesis [12][13][14][15][16][17]. However, the effects of Plasmodium infection on HCC metastasis and recurrence especially in tumor-resected animals remains unclear. Furthermore, there are no any data regarding the possible effects of Plasmodium infection on EMT pathways.
Here, we investigated the antitumor response of Plasmodium infection in progression, recurrence and metastasis of HCC in murine orthotopic resection model and non-resection model. We found that Plasmodium infection signi cantly inhibited growth, recurrence and metastasis of HCC and prolonged the survival of tumor-bearing mice in both models. Furthermore, signi cantly higher expression levels of E-cadherin and lower expression levels of Snail were observed in Plasmodium treated group compared with those in control group. Moreover, the activation of AKT and GSK-3β and the expression of CCR10 were suppressed by the infection. These ndings suggest that Plasmodium infection inhibits progression, recurrence and metastasis of HCC in tumor resection and non-resection models potentially via suppression of CCR10-mediated AKT/GSK-3β/snail signaling, which provides a possible new mechanism of action for further research.

Ethics statement
The animal experiment facilities were approved by the Guangdong Provincial Department of Science and Technology and complied with the guidelines of the Animal Care Committee, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. According to the international regulations, the mice were killed in time within the legal tumor size. All efforts were made to minimize animal suffering.

Sources of animals, cells, and parasites
Six to eight-week old female C57BL/6 mice purchased from Vital river Experiment Animal Limited Company (Beijing, China) were bred in the Animal Center of Guangzhou Institute of Biomedicine and Health GIBH following the Guide to the Care and Use of Laboratory Animal Committee of the institute. Mice were housed in speci c pathogen-free (SPF) conditions, with a 12-hours light cycle and food and water at ad libitum. All animal experiments were performed with the standard guidelines for the care of animals, which were approved by the Welfare Committee of the Center of Experimental Animals (Guangzhou, China). The Hepa1-6 Luciferase cell line, a murine liver cancer cell line derived from C57BL/6 mice was obtained from our deposit at GIBH and was incubated in a humidi ed atmosphere of 5% CO2 at 37°C. Plasmodium yeolii nonlethal strain (Py17XNL) was obtained from the Malaria Research and Reference Reagent Resource Center (MR4).

Animal models
To better understand the effect of Plasmodium infection on HCC recurrence and metastasis, two animal models were established as follows. Tumor volumes were calculated as follows: tumor volume = (length*width 2 )/2.
The non-resection model: To determine the effect of Plasmodium infection on HCC metastasis, the nonresection mouse model was established by intrahepatic injection of 5×10 5 Hepa1-6 Luciferase cells. Female C57BL/6 mice (n=56) were randomly divided into two groups, with 28 in each group. Control group (Hep) were only seeded with 5×10 5 Hepa1-6 Luciferase cells. Plasmodium infected group (Hep + Py) were orthotopically implanted with 5×10 5 Hepa1-6 Luciferase cells and intraperitoneally injected with The resection model: To investigate the effect of Plasmodium infection on post-operation recurrence, the resection mouse model was established by intrahepatic injection of tumor pieces and undergoing tumor resection on day 21 after tumor implantation. Tumors were derived from subcutaneous tumor-bearing mice, which was established by subcutaneous injected with 5 × 10 5 Hepa1-6 Luciferase cells. Tumors were harvested and cut into 1mm 3 pieces on day 14 post inoculation. The tumor pieces were implanted into the livers of 20 mice and Luciferase in vivo imaging was used to monitor the surviving of transplanted tumors in the following days. These mice underwent reoperation on day 21 post inoculation to completely remove the transplanted tumor mass. Then the mice were randomly divided into 2 group according to the inoculation with or without the parasite on day 3 after surgery. Twenty mice (10 each group) were used for the observation of survival and intraperitoneal metastasis of hepatocellular carcinoma.

Luciferase in vivo imaging
To evaluate tumor survival, we used the luminescence properties of luciferase to track HCC in vivo. In the trial, each mouse was injected intraperitoneally with potassium uorescein as a substrate according to body weight (10 ul/g uorescein potassium each mouse). IVIS Spectrum was used to monitor the uorescence emitted by Heap1-6 Luciferase about 10 to 20 minutes later.

Quantitative real-time PCR
Total RNA of tumor tissues were extracted with TRIzol reagent (Invitrogen Cat#15596018) and the RNA was reverse transcribed with the use of cDNA synthesis kit (TAKARA Cat#RR047A). Quantitative real-time PCR reactions were performed using TB Green Premix Ex Taq (TAKARA Cat#RR820A). The mRNA levels of the genes of interest were normalized to that of Actin. The Primer sequences for RT-qPCR were as follows: Actin: forward 5′-TCTGGCACCACACCTTCTAC-3′ and reverse 5′-TCATCTTTTCACGGTTGGCCT-3′, CCR10: forward 5′-CAAGCCCACAGAGCAGGTCTC-3′ and reverse 5′-GATCGGGTAGTTCGTCTGGC-3′, each sample was tested at least three independent replicates.

Statistical analysis
Statistical analysis was performed using GraphPad Prism. Results are presented as means ± standard deviations. A two independent sample t test was applied to compare means of two groups. One-way ANOVA was applied to compare means of multiple groups. Chi-square test was applied to compare the rate of metastasis of two groups. A P-value lower than 0.05 was considered statistically signi cant. Pvalue less than 0.05, 0.01, and 0.001 were indicated by *, **, and *** respectively, in each gure.

Results
Plasmodium infection signi cantly inhibited HCC progression and metastasis in the non-resection mouse model To investigate the effect of Plasmodium infection on the growth of HCC, mice were orthotopically seeded with Hepa1-6 luciferase cells and at the same time infected with Plasmodium yoelli 17XNL (Hep + Py) or uninfected with the parasite (Hep). In this experiment, Luciferase in vivo imaging was used to examine the viability of liver cancer cells, since the uorescence could be released by Hepa1-6 luciferase cells once combined with the substrate. The imaging results showed that all mice were successfully implanted with Hepa1-6 cells and the uorescence signal was signi cantly lower in Plasmodium treated group compared with that in the control group on day 15 post inoculation (Fig.1A). The peak of infection appeared around day 18 after intraperitoneal injection of the parasite in both models, and the highest parasite density reached about 55-60% on average and then fell back to 0 around 24-27 days ( Fig. S1A & B, & Fig. S2). The dynamics of parasitemia and self-healing of the infection were consistent with those in our previous studies [16]. There was no signi cant difference in body weight between the two groups during the course of Plasmodium infection (in the treated group), whereas the weight was incomparable between the two groups thereafter because most of the control mice died (Fig.1B). Importantly, the tumor-bearing mice treated with Plasmodium parasite showed better survival rate and longer overall survival compared with the control mice ( Fig.1C) (p<0.001). The tumor volume on day 15 after inoculation in Plasmodium treated group was signi cantly smaller than that in the control group (Fig.1E&F) (p<0.001). The tumor weight on day 15 after inoculation in Plasmodium treated group was also signi cantly lower than that in the control group (Fig.1G&H) (p<0.001). In addition, we found that there was almost no metastasis in the intraperitoneal of Plasmodium treated group, while there were many intestinal and abdominal metastases in the control group (Fig.1D) (p<0.001).

Plasmodium infection remarkably suppressed HCC recurrence in the resection mouse model
To characterize the effect of Plasmodium infection on HCC recurrence, the resection mouse model was established by intrahepatic implantation of tumor pieces and undergoing operation to completely remove tumors on day 21 after tumor implantation ( Fig.2A). In the early stage of Plasmodium infection, there was no signi cant difference in weight between the two groups. After resection of tumor, weight loss and short-term weight recovery occurred in both groups. During the period of infection, the diet of mice in Plasmodium infection group was poor, thus the weight growth in Plasmodium infection group was slower than that in the control group. After self-healing of infection, the weight in Plasmodium infection group continued to increase, while that of the control group decreased due to tumor development (Fig.2B). An effect of surgical resection followed by Plasmodium infection on weight loss was apparent while compared the infected group in resection model with the infected group in non-resection model ( Fig.2B and Fig.1B). A phenomenon of hemozoin accumulation in the liver (dark color) [18] was found in Plasmodium infected group in either resection model (Fig.2D) or non-resection model (Fig.1E). Importantly, our results demonstrated that Plasmodium treated tumor-bearing mice survived much longer than the control mice (Fig.2C). The cumulative recurrence rate in Plasmodium infection group was much lower than that in control group on the 75 th day (Fig.2D) (p<0.001).

Plasmodium infection inhibited the metastasis of HCC potentially through suppression of EMT
The EMT of HCC cells is closely associated with its ability of metastasis. E-cadherin is the key cadherin to prevent tumor metastasis and the most important biomarker of EMT, which is also crucial for HCC cells to maintain the normal cytoskeleton and the connection between homotypic cells [5]. Expectedly, the expression level of E-cadherin in Plasmodium treated group was signi cantly higher than that in control group (Fig.3, left & middle). The Snail superfamily of Zinc-nger transcription factors is a crucial transcription inhibitor for EMT, which can directly lead to inhibition of E-cadherin expression. As one of the biomolecules of E-boxes of the E-cadherin promoter, Snail could directly down-regulate the expression of E-cadherin and promote the occurrence of EMT [19]. Our results indicated that the expression of Snail showed a signi cant decrease in Plasmodium-treated group compared with control group (Fig.3, left & right).

Plasmodium infection suppressed the activation of PI3K/Akt/GSK-3β signaling
Previous studies have indicated that the PI3K/Akt/GSK-3β signaling and the transcriptional repressor Snail were required for EMT of HCC [9,11]. Activated downstream molecules of PI3K/Akt/GSK-3β could mediate the stabilization of endogenous Snail. Therefore, we tested if Plasmodium infection had an in uence on PI3K/Akt/GSK-3β pathway in HCC cells. Western blot analysis showed that the phosphorylation of Akt (Fig. 4A&C) (p<0.05) and GSK-3β (Fig. 4A&D) (p<0.05) in Plasmodium-treated group was downregulated, as compared with that in control group, even though the difference of PI3K lacked statistical signi cance between the two groups (Fig. 4B). These ndings suggested that Plasmodium infection could inhibit the activation of PI3k/Akt/GSK-3β signaling pathway, thus suppressed the accumulation of Snail and EMT programing.

Plasmodium infection downregulated the expression of CCR10
Chemokines/chemokine receptors axis have been demonstrated to have the ability to activate PI3K/Akt/GSK-3β/Snail signaling pathways in HCC cells [9,11]. To speci cally address the role of CCR10 mediated-PI3K/Akt/GSK-3β/Snail signaling in Plasmodium infection, we examined the expression of CCR10 in tumor tissues derived from tumor-bearing mice. As expected, the mRNA expression level of CCR10 was signi cantly lower in Plasmodium treated group than that in control group (Fig.5B) (p<0.01).
Western blot result showed that the expression level of CCR10 was also signi cantly lower in the infected group than that in the control group. (Fig.5A) (p<0.05).

Discussion
Our present study indicates that Plasmodium infection signi cantly prolongs the survival time of tumorbearing mice in both HCC models through inhibiting the tumor growth and abdominal metastasis. The underlying mechanisms of action might involve the inhibition of PI3K/Akt/GSK-3β/Snail signaling by down-regulating the expression of CCR10, reduction of Snail accumulation, and up-regulation of Ecadherin.
Primary hepatocellular carcinoma ranks the fourth in the cause of cancer-related death worldwide. Although surgical resection can remove the visible tumor, it cannot cure liver cancer. Hepatoma cells can escape from the microenvironment and pass through blood vessels and lymphatics, which can lead to tumor recurrence and metastasis. This malignant behavior is the leading cause of postoperative death in patients with liver cancer [20]. Therefore, how to prevent metastasis of liver cancer cells and to reduce postoperative recurrence are the key element to improve the prognosis of patients with liver cancer. Our current study indicates that Plasmodium infection signi cantly reduces the postoperative recurrence and metastasis in the murine HCC resection model.
The epithelial-mesenchymal transition (EMT) has been implicated in carcinoma invasion and metastasis and is the initial step for epithelial cancer cells to escape from homologous cells [21]. Previous studies have suggested that down-regulation of E-cadherin is a prominent feature of EMT. It has been demonstrated that the transcriptional activity and protein expression of E-cadherin can be enhanced by preventing the binding of E-boxes of the E-cadherin promoter to transcriptional barriers (including Zincfinger transcriptional repressors snail and slug, the repressor SIP-1/ZEB-2, DEF-1/ZEB-1, as well as the basic helix-loop-helix transcription factors twist and E12/E47), which prevent EMT [21,22]. Our current study indicates that Plasmodium infection increased the expression of E-cadherin and downregulating Snail, which suggests that Plasmodium infection might prevent EMT in HCC.
PI3K activates and produces second messenger PIP3, which activates AKT through phosphorylation of AKT protein at Ser308 [23]. GSK-3β is a downstream target of phosphorylated AKT, which promotes the degradation of Snail, while phosphorylation of GSK-3β leads to its inactivation. Phosphorylated AKT can change GSK-3β into phosphorylated GSK-3β, which leads to the inactivation of GSK-3β and the increase of Snail. Snail, as a transcription inhibitor of E-cadherin, is the downstream factor of GSK-3β. GSK-3β inhibits the expression of Snail and increases E-cadherin protein expression [24]. Our current study indicates that Plasmodium infection inhibits the phosphorylation of AKT, which inhibits the transition from GSK-3β to phosphorylated GSK-3β, and reduces the expression level of Snail protein. Finally, the inhibition effect of Snail on E-cadherin is removed and EMT might be prevented.
Chemokines are known as small (8 to14 kDa) chemoattractant cytokines that selectively regulate the recruitment and tra cking of leukocyte subsets to the sites of in ammation. According to the spacing of the rst two cysteines in the N-terminus, chemokines are divided into several subfamilies including C, CC, CXC, and CX3C chemokines. Chemokines exert its action through seven trans-membrane spanning Gprotein-linked receptors (chemokine receptors) [25,26]. Previous studies have suggested that chemokines and chemokine receptors are involved in in ammatory reactions and wound healing [27]. Recently, researches demonstrated that chemokines and their receptors play a critical role in tumor cell growth and metastasis in melanoma, lung cancer, gastric carcinoma, pancreatic cancer, colorectal carcinoma and hepatocellular carcinoma [28]. It has been reported that CCR10 activation stimulates the progression, invasion and migration of HCC, breast cancer cells and melanoma cells [11,29,30]. CCR10 expression and downstream PI3K/Akt pathway shows a role in HCC progression [11]. Our results showed that Plasmodium infection could downregulate the expression of CCR10. The inhibition of CCR10-mediated PI3K/Akt/GSK-3β/Snail signaling might result in suppression of EMT programing in HCC.

Conclusions
In summary, our ndings in current study might involve a possible new mechanism (Fig. 6

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional les.

Competing interests
Authors declare no con ict of interest.

Funding
This study was supported by the