Tumor growth and metastasis can be inhibited by maintaining genomic stability in cancer cells

Abstract

The existence of cancer stem cells, stem-like cancer cells (SLCCs), or tumor-initiating cells is considered as the cause of tumor formation and recurrence, indicating the importance of studying novel therapy that targets SLCCs. The origin of SLCCs is controversial because of two competing hypotheses: SLCCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Our previous research demonstrates that SLCCs are inducible by increasing genomic instability in cancer cells. In this study, to block the emergence of SLCCs, aminoethyl isothiourea (AET), a compound that clears free radicals and is used to protect patients from radioactive exposure, was used as an agent that maintains genomic stability in combination with mitomycin C (MMC), a commonly used chemotherapeutic drug that damages DNA. Using a rabbit tumor model with VX2 hepatic carcinoma, we found that MMC alone increased lung metastases and disadvantaged survival outcome, but the combination of MMC and AET reversed this effect and even prolonged overall survival. Moreover, in a VX2 xenograft model by immunocompromised mice, MMC alone enriched tumor-initiating cells, but the administration of MMC in combination with AET eliminated tumor cells effectively. Furthermore, MMC alone enhanced genomic instability, but MMC combined with AET attenuated the extent of genomic instability in primary VX2 tumor tissue. Taken together, our data suggest that the genomic protector AET can inhibit the induction of SLCCs, and this combination treatment by AET and cytotoxic agents should be considered as a promising strategy for future clinical evaluation.

Keywords

rabbit VX2 liver tumor / mitomycin C / AET / stem-like cancer cells / genomic instability

Introduction

The existence of cancer heterogeneity has been widely acknowledged. Genomic instability is a hallmark of cancer, which is considered as the main reason for cancer heterogeneity [ 1]. Recent studies suggest that in tumor tissue, a subset of cells harbor stem cell-like features that promote self-renewal and maintain tumor growth and hierarchy [ 2]. This subpopulation of cells is referred to as cancer stem cells, stem-like cancer cells (SLCCs), or tumor-initiating cells [ 3, 4]. As demonstrated by many publications, SLCCs have a strong ability of tumorigenesis and contribute to the resistance of some individuals to cancer therapy [ 5]. Therefore, SLCCs are responsible for tumor recurrence. Furthermore, SLCCs may act as an important contributor for invasion and metastasis because of their strong self-renewal and tumor-initiating ability as well as the tendency to undergo epithelial-mesenchymal transition, which can endow themselves with notable motility, invasiveness, and heightened resistance to apoptosis [ 6, 7]. Taken together, studying the association between genomic instability and the origin of SLCCs, as well as the relevant targeted therapy against SLCCs, is significant.

We have documented that genomic instability can contribute to the origin of SLCCs [ 4]. Conventional chemotherapeutic agents (e.g., mitomycin C (MMC)) are cytotoxic usually because they initiate DNA damage, which is a common inducer of genomic instability. Thus, although chemotherapies alone may kill cancer cells temporarily, enrichment of SLCCs follows and eventually leads to relapse or metastasis. If this is the case, targeting genomic instability simultaneously is necessary when chemotherapy is applied. Therefore, we hypothesize effective eradication of cancer cells by the combination of conventional chemotherapeutic drugs and compounds that maintain genomic stability because this novel chemotherapeutic strategy does not only destroy “common” cancer cells but also blocks the emergence of SLCCs, resulting in the prevention of relapse or metastasis.

Aminoethyl isothiourea (AET), a compound that clears free radicals, was developed to protect patients from radioactive exposure [ 8, 9], and currently is a cure for coma [ 10]. In this study, AET is used as the agent that maintains genomic stability in combination with MMC, a commonly used chemotherapeutic drug that damages DNA in cancer cells.

Materials and methods

Animal studies

All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Sun Yat-sen University Cancer Center Animal Care and Ethics Committee. To generate the VX2 liver tumor model, New Zealand rabbits with body weight of approximately 2 kg were obtained from the Animal Experimental Center of Sun Yat-sen University (Guangzhou, China). Fragments of VX2 tumor tissue (stored in our laboratory) with a size about 0.5 mm³ were transplanted into the liver lobes of rabbits by puncture needle in a surgical operation. Two weeks after transplantation, the rabbits were randomly divided into 3 groups, including NS, MMC, and MMC+ AET (10 animals per group), and intravenously treated with 1 ml/kg normal saline (NS, 0.9% NaCl), 0.75 mg/kg MMC, and 1.7 mg/kg AET (Sigma-Aldrich). These treatments were administered once a week and lasted for four weeks. When the rabbits became weak and exhausted, they were humanely euthanized and anatomized. For each animal, the entire liver and lung were measured and photographed, and samples of primary liver tumor were collected for further tissue dissociation, DNA extraction, and histological assessment. The volume of primary liver tumors was calculated using the formula V= 0.5×(length×width²).

For the tumorigenic assays in the xenograft model, 4- to 6-week-old female athymic nude mice were obtained from the Animal Experimental Center of Sun Yat-sen University Cancer Center. The primary VX2 liver tumors were chopped into fragments and dissociated by 0.5 mg/ml collagenase solution (Type IV, Sigma-Aldrich) for 2 hours at 37 °C. Thereafter, the mice were given subcutaneous injections of 3×10⁵, 1×10⁶, and 3×10⁶ tumor cells in their left and right axillary areas. The animals were monitored twice a week for two months before humane sacrifice.

Copy number variation (CNV) analysis

Samples of three cases of primary VX2 liver tumors from each group were collected, and DNA was isolated by using a NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer’s instructions. After amplification, extracted DNA from each group was pooled and hybridized on NimbleGen DNA Microarrays (Roche) following the manufacturer’s protocol. Using the hybridization data of normal rabbit liver tissue as control, the copy number variation of VX2 liver tumors was calculated and analyzed by Segmentation algorithm of Partek Genomics Suite. Experimental process and data analysis were performed by CapitalBio Corp. in Beijing, China.

Statistical analysis

The data were presented as mean±SD except where indicated. Comparisons between two groups were subjected to 2-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way ANOVA with Bonferroni post hoc test. For survival analysis, survival curves were obtained using Kaplan-Meier method, and the pairwise multiple comparisons were conducted by Holm-Sidak method. Data processing was performed using SPSS 16.0 and SigmaPlot 11.1. P<0.05 was considered to be statistically significant.

Results

MMC alone disadvantages outcome, but the combination of MMC and AET promotes overall survival

To observe the efficacy of our treatment in vivo, we applied rabbit VX2 liver tumor model in this study. The VX2 carcinoma, derived from a virus-induced papilloma of rabbits in the 1940s [ 11], has been extensively used to study different aspects of tumor behavior and is generally accepted for the establishment of a liver tumor [ 12, 13]. In the condition without any treatment, VX2 liver tumors in rabbits grew so fast that the animals became weak and exhausted 6–10 weeks after inoculation, and eventually none of the rabbits survived in the long term. After humanely sacrificing the weak tumor-bearing rabbits, we performed detailed necropsies followed by pathological validation (data not shown), and usually found aggressive liver tumors accompanied by ascites and/or lung metastases. Interestingly, the survival situation of the MMC group was worse than that of the vehicle control (NS) group, whereas the combination of MMC and AET dramatically prolonged overall survival, and long-term surviving cases were even found in the MMC+AET group (Fig. 1A). The median survival time was 47 days for the NS group, 35 days for the MMC group, and 83 days for the MMC+AET group, respectively. Pairwise multiple comparisons showed that the differences were statistically significant (Fig. 1B).

MMC alone increases lung metastases but MMC and AET reverse this effect

In the vehicle control group, tumor-bearing animals were only treated with normal saline, which usually results in a large bulk of primary liver tumor. By contrast, for the MMC and MMC+ AET groups, the mean sizes of primary liver tumors were significantly decreased compared with that of the vehicle control group (NS vs. MMC, P<0.001; NS vs. MMC+ AET, P<0.001), which indicates the effective cytotoxicity of MMC (Fig. 2A and 2B). However, we found that the mean wet weight of lungs for the MMC group was 76.9 g, which is significantly higher than that of the vehicle control group (40.8 g, Fig. 2C), suggesting that lung metastases were promoted by the treatment of MMC alone, which may consequently cause the unfavorable outcome in the MMC group (Fig. 1A and 1B). Notably, lung metastases seldom happened in the animals in the MMC+ AET group (Fig. 2A), and the mean wet weight of lungs for this group is only 17.6 g, dramatically less than that of the MMC group (P<0.001, Fig. 2C). These data showed that the combination of MMC and AET repressed both primary liver tumors and lung metastases, consistent with the favorable overall survival of the MMC+ AET group (Fig. 1A and 1B).

MMC alone enriches tumor-initiating cells, but MMC and AET effectively eliminate tumor cells

To test the hypothesis that MMC alone could enrich SLCCs whereas the combination of MMC and AET reversed this effect, we observed the impact of MMC and AET on the self-renewal capacity of VX2 liver tumor cells in our models. Since VX2 tumor cells could not be cultured in vitro, we applied xenotransplantation assays in nude mice to assess the tumorigenic potential of primary VX2 liver tumor cells. Six weeks after 3×10⁵ or 1×10⁶ cells were transplanted, only the tumor cells from the MMC group produced xenografts. When the cell number increased to 3×10⁶, the MMC-treated tumor cells formed xenografts in 5 of 8 mice, whereas the NS-treated tumor cells from the vehicle control group generated xenografts in 3 of 8 mice. Interestingly, for the MMC+AET group, no xenograft could be produced at any transplanted cell dose of primary liver tumors (Table 1). These observations showed that the primary liver tumor cells of the MMC group had highly strong tumorigenic ability while those of the MMC+AET group were hardly tumorigenic in vivo, which is consistent with the extremely metastatic potential of the MMC-treated liver tumor cells.

MMC alone enhances genomic instability, but MMC and AET attenuate the extent of genomic instability

To evaluate the extent of genomic instability induced by MMC and AET, we performed rabbit-specific microarray comparative genomic hybridization (array-CGH) assays and checked the genome-wide CNV in VX2 tumor cells. As shown in Table 2, compared with normal liver control, the primary liver tumor cells of the MMC group harbored 1396 of copy number (CN)-altered loci (611 for amplification and 785 for deletion), much more than the counterparts in the vehicle control group (a total of 1035 CN-altered loci), indicating that the treatment of MMC alone promoted genomic instability of the primary liver tumor cells. By contrast, for the MMC+ AET group, the primary liver tumor cells had fewer CN-altered loci (300 for amplification and 514 for deletion, 814 in total) than those in the vehicle control group, suggesting the decreased extent of genomic instability led by the addition of genome protector AET. Using segmentation algorithm to analyze the hybridization data, we obtained similar results, showing that the primary liver tumor cells of the MMC group owned more CN-altered segments than those of the vehicle control (476 vs. 356, Table 2). Moreover, the number of CN-altered segments in the MMC+ AET group markedly declined compared with that in the vehicle control (259 vs. 356, Table 2), consistent with the data on CN-altered loci number. We further analyzed all the detected CNV loci of the three groups of cancer cells (Supplementary File 1), and determined the differences and similarities of altered CNV loci among them. As shown in Fig. 3, all the three groups shared 448 CN-altered loci, and the MMC group had 474 unique CN-altered loci, much more than that in the vehicle control (150) and the MMC+ AET group (133). Taken together, all these results indicated that MMC alone enhanced genomic instability, but MMC and AET attenuated the extent of genomic instability.

Discussion

In this study, we validated our previously proposed theory that increasing genomic instability initiated by DNA damage in cancer cells could induce the generation of SLCCs [ 4] by using a large animal model of cancer, and further designed a novel SLCC-targeted chemotherapeutic strategy, which is a combination of one DNA-damaging cytotoxic drug and the compound that maintains genomic stability. This strategy managed to repress metastasis, benefit survival outcome, and eliminate SLCCs in rabbit VX2 liver tumor models.

MMC is a potent DNA crosslinker that can block DNA replication and subsequently trigger replication arrest and cell death if the crosslink is not repaired. It has a wide clinical anti-tumor spectrum with efficacy in various tumor types such as gastric, pancreatic, bladder, breast, and liver cancer [ 14]. However, such conventional chemotherapeutics, which efficiently target actively proliferating cells within the primary tumor, have a minimal impact on quiescent or slowly proliferating cancer cells and, thus, on cells that reside in many micrometastatic colonies [ 7]. Consistently, we observed a dramatically decreased proliferation of primary liver tumors rather than repressed lung metastasis in the MMC group compared with the vehicle control group (Fig. 2B and 2C). Furthermore, considering the chemoresistant trait of SLCCs [ 5], we observe that chemotherapy selectively enriches SLCCs, which has been proved by previous reports [ 4, 15] and our current data (Table 1). In this case, based on the plausible connection between SLCCs and cancer metastasis, chemotherapeutic drugs may play a part in facilitating cancer metastasis; this presumption is supported by our observation that MMC alone increases lung metastases (Fig. 2C).

AET acts as a strong radical scavenger in which the sulfhydryl groups compete successfully with radiation-sensitive biological molecules for the radicals generated when ionizing radiation decomposes water [ 16]. Possibly, AET may protect by combining reversibly with biologically important molecules such as enzymes [ 17]. Therefore, AET has been used as an effective radioprotective agent, as evidenced by previous in vivo studies in mice and primates [ 18, 19]. Thus, in this study, we applied AET as a genome protector in rabbit tumor models, combined with the conventional anti-tumor drug MMC, which can induce DNA damage and genomic instability. Since the intravenous administration of AET with a dose of 1.7 mg/kg shows no cytotoxic efficacy in our pilot experiment (data not shown), we did not set up an AET-alone group. Our results for CNV detection show that AET facilitates the decrease of the extent of genomic stability (Table 2) and blocks the emergence of SLCCs (Table 1) in the case of the MMC+ AET combination. However, given the potential correlation between the ability of MMC to generate reactive oxygen radicals and its cytotoxicity to tumor cells [ 20], we cannot exclude the possibility that partial cytotoxic activity of MMC might be counteracted by the presence of AET, although no difference exists in primary liver tumor volume between the MMC and MMC+ AET groups according to our current data (Fig. 2B). Meanwhile, because of the significant efficiency of radioprotection of AET [ 18, 19], AET possibly facilitates the function of DNA damage response and repair in VX2 tumor cells, which may also partially counteract the cytotoxic activity of MMC. To further unveil the underlying mechanism of AET’s suppressive activity against SLCCs, we have to conduct more detailed investigations in the future.

In summary, this study verifies the intrinsic association between the genomic instability induced by DNA damage and the origin of SLCCs by large animal experiments. It also documents for the first time the application of AET that can maintain genomic stability as a successful inhibitor against the emergence of SLCCs. Our results show that the combination of MMC and AET eliminates VX2 liver tumor cells effectively, decreases lung metastases, and improves overall survival, which implies that future clinical cancer therapy should not only target common cancer cells, but also prevent SLCC formation by maintaining genome stability.

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