Overexpression of CIP2A is associated with poor prognosis in multiple myeloma

Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous protein phosphatase 2A (PP2A) inhibitor, has been identified as an oncoprotein in promoting cancer initiation and progression of several types of cancer. However, the expression and the role played by CIP2A in the pathogenesis of multiple myeloma (MM) remain unclear. In this study, we showed that CIP2A was overexpressed in human MM cell lines and MM patients’ bone marrow tissues. Clinicopathologic analysis showed that CIP2A expression was significantly correlated with clinical stage and percent of plasma cells in bone marrow. Kaplan–Meier analysis revealed that patients with high CIP2A expression presented with poorer overall survival rates than those with low CIP2A expression. Moreover, CIP2A knockdown in MM cells resulted in attenuated proliferative abilities. In addition, CIP2A depletion sensitizes dexamethasone (Dex)-resistant cells to Dex. The effect of CIP2A on proliferation and Dex therapy was mediated by the inhibition of PP2A, which in turn activated Akt. In vivo studies confirmed that CIP2A regulated MM tumorigenesis and the phosphorylation of Akt. Taken together, our results suggest that CIP2A oncoprotein plays an important role in MM progression and could serve as a prognosis marker and a novel therapeutic target for the treatment of patients with MM.


INTRODUCTION
As a neoplastic disorder of plasma cells characterized by clonal proliferation within the bone marrow (BM), multiple myeloma (MM) accounts for~10% of all hematological cancers and~1% of all cancer deaths. 1 In 2017, the annual incidence of MM increased with the aging of the population worldwide. An estimated 30 280 new cases (17 490 in men and 12 790 in women) of MM were diagnosed in 2017, and 12 590 deaths (6660 in men and 5930 in women) are estimated to occur from this disease in the United States. 2 Overall survival has improved in recent years. Patients are now predicted to have a median survival of~5 years. 3 Unfortunately, despite the improved therapeutic armamentarium, nearly all patients experience relapse and refractory disease. To date, MM remains an incurable disease. Therefore, there is an urgent need to explore novel therapeutic target and agent to improve the survival of patients with MM.
The oncogenic transformation of human cells requires the perturbation of a distinct set of oncogenes and tumor suppressors. Protein phosphatase 2A (PP2A) complexes function as tumor suppressors by inhibiting the activity of several critical oncogenic signaling pathways such as Akt and ERK. 4 Consequently, inhibition of PP2A phosphatase activity is one of many prerequisites for the transformation of normal human cells into cancerous cells. However, the mechanisms underlying PP2A inactivation in human cancers are poorly understood. Cancerous inhibitor of protein phosphatase 2A (CIP2A), a recently identified endogenous PP2A inhibitor in malignant cells, is one possible mechanism. CIP2A stabilizes c-Myc protein by inhibiting its degradation mediated by PP2A in cancer cells. 5 In addition to inhibiting c-Myc degradation, CIP2A is regulated in a positive feedback loop with c-Myc by promoting each other's expression. 6 Previous independent studies showed that aberrant overexpression of CIP2A is associated with tumor growth, resistance to apoptosis, drug resistance, prognosis and metastasis in many human solid malignancies including head and neck, 5 gastric, 7 breast, 8 tongue, 9 ovarian, 10 and lung cancers. 11 Besides, CIP2A downregulation and inactivation of the Akt pathway have been reported to inhibit the proliferation and to induce apoptosis in a variety of hematologic neoplasms. [12][13][14] Importantly, recent studies provided evidence that high CIP2A expression is a poor prognostic factor in normal karyotype acute myeloid leukemia (AML). 12 Barragán et al. 12 showed that high CIP2A expression is a recurrent event in AML, where it represents a marker of reduced overall survival and a poor prognostic factor, as previously reported in other tumors. In addition, CIP2A depletion downregulates cell proliferation. Thus, CIP2A represents a novel therapeutic target in AML. Furthermore, CIP2A is associated with radioresistance and modulates the sensitivity of breast cancer cells to bortezomib treatments. 15 Choi et al. 16 determined that CIP2A expression is associated with the sensitivity to doxorubicin and CIP2A overexpression in MCF-7 cells overcame the inhibition of cell proliferation in response to doxorubicin treatment. Moreover, CIP2A knockdown may sensitize metastatic castration-resistant prostate cancer cells to cabazitaxel chemotherapy. 17 Liu and colleagues 18 also showed that CIP2A knockdown increased the drug sensitivity of HeLa and doxorubicin-resistant HeLa cells. In addition, CIP2A is involved in regulating multidrug resistance of cervical adenocarcinoma upon chemotherapy by enhancing P-glycoprotein expression through E2F1. Conclusively, De and colleagues 1 reviewed that CIP2A acts as an 'oncogenic nexus' contributing to cancer development, cancer evolution and drug resistance, suggesting that CIP2A can be targeted in cancer. However, the role of CIP2A in MM tumorigenesis and metastasis remains less elusive. Recently, Yang et al. 19 determined that CIP2A silencing inhibited proliferation and promoted apoptosis in MM cell lines RPMI 8226 and NCI-H929 cells.
In this study, we analyzed the expression of CIP2A in MM specimens and cell lines by real-time quantitative PCR, western blot and immunohistochemistry. We also measured the effect of CIP2A on MM cell proliferation and dexamethasone (Dex)mediated inhibition of cell proliferation in Dex-resistant cell lines. Our findings uncover the mechanisms of CIP2A-mediated tumorigenesis and Dex therapy.

MATERIALS AND METHODS Patients
MM patient's samples were obtained from the Department of Oncology, the Second Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China); Department of Pathology, Zhuhai Hospital Affiliated to Jinan University (Zhuhai, China); and Department of Hematology, Nanfang Hospital Affiliated to Nanfang Medical University (Guangzhou, China) after informed consent. Forty-one patients were studied. The diagnosis was based on clinical data and examination of BM according to French-American-British classification. CD138 + cells from 11 MM patients were isolated with informed consent from BM mononuclear cells using positive immunomagnetic column separation (Miltenyi Biotech, Auburn, CA, USA). The purity of CD138+ cells is above 97% as determined by flow cytometry. Peripheral blood mononuclear cells from four healthy donors were separated by Ficoll-Hipaque density sedimentation. 20 Real-time quantitative PCR was used to detect these 11 MM patients and 4 healthy donors. Forty-one MM patients' BM and 22 adjacent normal bone trabecula and BM paraffin section were detected by immunohistochemical analysis.

Cells and reagents
The human MM cell lines U266 and RPMI 8226 (8226), and the promyelocytic cell line HL-60 were purchased from American Type Culture Collection (Manassas, VA, USA). Dex-sensitive (MM.1S) and Dex-resistant (MM.1R) human MM cell lines were kindly provided by Professor Jie Jin (Department of Hematology, The First Affiliated Hospital, Zhejiang University College of Medicine). All cell lines were incubated in a humidified CO 2 incubator (5% CO 2 , 37°C).
Transfection of DNA, siRNA and stable cell line generation The pOTENT-1-CIP2A expression plasmid was purchased from Youbio Co. (Changsha, China). Transfection of the pOTENT-1-CIP2A plasmid into MM cells were carried out using Lipofectamine 3000 transfection reagent (Invitrogen) following the manufacturer's protocol.

Cytotoxic assay and cell viability
Cells were seeded into a 96-well plate and precultured for 24 h, and then transfected with plasmid or siRNA for 24 or 48 h. Cell cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) or CCK-8 assay. Cell viability was estimated by trypan blue dye exclusion. 18 PP2A phosphatase activity PP2A immunoprecipitation phosphatase assay kit (Upstate, Temecula, CA, USA) was used to measure phosphate release as an index of phosphatase activity, according to the manufacturer's instructions. 23 Flow cytometric assays for Annexin-V Cell apoptosis was evaluated by the Annexin-V detection using an AV-FITC kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions. 20 Murine model All mice used in this study were bred and maintained in a specific pathogen-free environment. BALB/c nude mice (6-7 weeks old) were . The mice were injected subcutaneously with 1 ×10 7 U266 (n = 7), CIP2A KD -U266 (n = 8), 8226 (n = 7) or CIP2A OE -8226 (n = 8) cells in 100 μl RPMI-1640 media into the right flank. Caliper measurements of the longest perpendicular tumor diameters were performed twice a week to estimate the tumor volume, using the following formula: 4π/3 × (width/2) 2 × (length/2), representing the three-dimensional volume of an ellipse.
Animals were killed when tumors reached 1.5 cm or if the mice appeared moribund to prevent unnecessary morbidity to the mice. At the time of the animals' death, tumors were excised; cells were separated and subjected to PP2A activity analysis or lysed for western blot.

Statistical analysis
All experiments were repeated at least three times and the data were presented as the mean ± s.d. unless noted otherwise. Differences between data groups were evaluated for significance using the Student's t-test of unpaired data or one-way analysis of variance and Bonferroni post test. For   (Figure 1c). In addition, CIP2A protein levels were detected by western blot. The results show that CIP2A protein expression is also higher and consistent with the messenger RNA expression levels in Dex-resistant MM.1R and U266 cells (Figure 1d). A previous study reported that the traditional chemotherapeutic agent doxorubicin downregulates CIP2A expression, and that increased CIP2A expression confers doxorubicin resistance in breast cancer cells. 16 Altogether, these data indicate that CIP2A is overexpressed in MM cell lines at the protein and messenger RNA levels. The higher CIP2A expression in Dex-resistant cells is worthy of further investigation.
As the MM cell lines presented higher CIP2A expression, we next sought to investigate whether patients with MM also express high levels of CIP2A. CIP2A expression was investigated by QPCR in 10 patients' CD138 + cells ( Figure 1e) and 4 healthy donors' peripheral blood mononuclear cells (Figure 1f). HL-60 cell line was also used as a positive control. The results indicate that CIP2A was expressed at relatively high levels in all patients, but expressed at relatively low levels in normal peripheral blood mononuclear cells from four healthy volunteers. Furthermore, we determined CIP2A expression in clinical samples using immunohistochemistry analysis in 41 MM specimens and 22 adjacent normal bone trabecula and BM tissues, and determined that CIP2A was overexpressed in 46.3% of the tumor samples (19 of 41), whereas most of the adjacent normal tissues exhibited undetectable or low CIP2A staining (Figures 1g and h). In 16 cases MM tissues with paired adjacent non-tumor tissues, we observed a significantly higher expression of CIP2A in tumor tissues compared with paired adjacent non-tumor tissues (P o 0.05, Figure 1i). These results indicated that CIP2A might be a critical molecule in MM development.
Furthermore, we analyzed the relationship between CIP2A expression levels and clinicopathological characteristics. As shown in Table 1, no significant correlation was observed between CIP2A expression and age, gender or type of myeloma (P40.05). However, both International Staging System stage (P40.05) and percent of BM plasma cells (P=0.045) show significant correlations with high level of CIP2A expression. Survival analysis revealed that MM patients with high CIP2A expression (n = 19) presented with a poorer overall survival than those with low CIP2A expression (n = 22; P = 0.033; Figure 1j) And, the median survival of low and high CIP2A groups were 66 and 45 months, respectively. Altogether, our data suggested that CIP2A is overexpressed in MM, and high levels of CIP2A expression are a prognostic predictor of progression and poor prognosis of patients with MM.
Effect of CIP2A expression on MM cell proliferation To identify whether an increase in CIP2A expression was associated with MM cell proliferation, MM.1S and 8226 cells were first transfected with CIP2A plasmid, and CIP2A expression was confirmed by using reverse transcription PCR and western blot (Figures 2a and b). CIP2A was highly expressed at both the    (Figures 3c and d). Furthermore, flow cytometry was used to detect apoptosis. The results suggested that CIP2A overexpression overcame the Dex-induced MM.1S cell apoptosis (P o 0.01, Figure 3e). In addition, CIP2A depletion enhanced the Dex-induced MM.1R cell apoptosis (P o 0.01, Figure 3f). To gain insights into the molecular mechanisms underlying the enhancing effect of reduced CIP2A expression on Dex effects, MM.1R cells were transfected with CIP2A siRNA, followed by Dex treatment. Western blot analysis was used to detect variations in the protein expression (Figure 3g). Interestingly, CIP2A silencing enhanced Dex-induced apoptosis. These results imply that CIP2A expression levels determine the sensitivity of MM cells to Dex.
Inhibition of PP2A is essential for CIP2A-induced proliferation and Dex therapy CIP2A participates in the regulation of molecular processes mostly by inhibiting the tumor suppressor PP2A. 5 Thus, we analyzed whether CIP2A deregulation can alter the effects of the tumor suppressor PP2A in MM cells. As expected, PP2A activation was observed after CIP2A silencing (P o 0.01, Figure 4a). Western blot analysis indicated that CIP2A silencing had no effect on PP2Ac (catalytic subunit) expression, but upregulated PP2A activity (Figure 4b). To evaluate whether CIP2A-induced cell proliferation results from the inhibition of PP2A activity, we compared cell proliferation in CIP2A siRNA-expressing MM.1R cells and MM.1R cells in the presence and absence of the PP2A inhibitor, okadaic acid (OA). Our results showed that OA treatment significantly abrogated the effect of CIP2A silencing on proliferation (P o 0.05, Figure 4c). Moreover, the sensitivity of CIP2Asilenced MM.1R cells to Dex was also markedly inhibited by OA (P o 0.05, Figure 4d). We next examined whether PP2A inhibition had any effects on the phosphorylation status of Akt, a target of PP2A. As expected, CIP2A knockdown decreased the phosphorylation of Akt without affecting its expression levels. Moreover, OA treatment rescued Akt phosphorylation in CIP2A knockdown U266 cells, as well as in CIP2A knockdown MM.1R cells (Figures 4e and f). Taken together, these findings suggested that PP2A inhibition is essential for CIP2A-induced proliferation and Dex therapy.

CIP2A promotes tumorigenesis in vivo
Because of the poor tumorigenicity of MM.1R and MM.1S cells, U266 and 8226 cells were used to establish the MM murine model to test the efficacy of CIP2A. 22 Two cell lines, CIP2A knockdown U266 (designed CIP2A KD -U266) and CIP2A overexpression 8226 (designed CIP2A OE -8226), were established respectively to further address the functional role of CIP2A in MM tumorigenesis in vivo. After 3 weeks of selection following Lipofectamine 3000 transfection, the CIP2A protein levels were confirmed by western blot (Figure 5a). Four groups of nude mice were subcutaneously inoculated into the right flank with U266, CIP2A KD -U266 cells, 8226 or CIP2A OE -8226, respectively. Animals were killed when the tumors reached 1.5 cm in diameter or when paralysis or major issues compromising in their quality of life occurred. After 1 month, the mice were killed and tumor tissues were isolated. Intriguingly, CIP2A KD -U266-transplanted mice presented fewer and smaller detectable tumor nodules compared to U266 transplanted mice (P o 0.05, Figures 5b and c). CIP2A OE -8226transplanted mice presented more and larger detectable tumor nodules compared to 8226-transplanted mice (P o0.05, Figure 5d). Consistent with these data, the survival of CIP2A KD -U266-transplanted mice was prolonged compared to that of U266-transplanted mice (Figure 5e). In addition, CIP2A depletion significantly reduced the tumor weight (P o0.05, Figure 5f), and CIP2A overexpression significantly increased the tumor weight (P o 0.05, Figure 5g). Tumors from mice bearing U266 or CIP2A KD -U266 cells were isolated, cells were collected and experiments were conducted to determine whether CIP2A silencing influenced its downstream molecules. As shown in Figure 5h, the level of Akt phosphorylation in CIP2A KD -U266 tumors was significantly decreased compared to that in U266 tumors. Meanwhile, consistent with the above-mentioned results, PP2A activity was significantly increased in CIP2A KD -U266 tumors (P o 0.05, Figure 5i). Altogether, these results indicated that blocking CIP2A/PP2A signaling could inhibit cell proliferation in MM.

DISCUSSION
In this study, the evidence supporting that CIP2A is associated with Dex therapy and poor prognosis in MM was proposed at the very first time. Previous reports indicated that the CIP2A gene is expressed at high levels in cancer cells from the B lymphocyte and myeloid lineages. 14,28, 29 Li et al. 29 used immunohistochemistry and QPCR to confirm that CIP2A is overexpressed in BM cells from patients with a group of high-risk myelodysplastic syndromes and might play a role in the proliferation of blasts in the myelodysplastic syndromes BM and in disease progression in at least some cases. In addition, Lilja et al. 30  Increasing evidence demonstrates that higher CIP2A expression promotes cancer progression, suggesting that CIP2A could be an attractive therapeutic target for the treatment of human cancers. 1,31 Next, we investigated the CIP2A expression status and its correlation with clinicopathological features of patients with MM. Our data showed that CIP2A expression was increased in MM patients' cells compared with that in adjacent normal bone trabecula and BM tissues (Figures 1e-i). We also demonstrated that higher CIP2A staining was significantly correlated with International Staging System stages, percent of BM plasma cells and worse survival in patients with MM ( Figure 1j; Table 1). These findings indicate that CIP2A may be involved in the progression of MM and could be a significant prognostic factor for patients with MM. Although more clinical trials are required to validate this finding, we hope that CIP2A can be used as a biomarker to improve the prognostic accuracy for patients with MM.
The clinical results urged us to carry out a series of in vitro and in vivo experiments to explore the potential mechanisms underlying the effects of CIP2A. In several types of solid cancer cells, CIP2A depletion leads to impaired proliferation, foci formation, cell migration, invasion and doxorubicin resistance. 6,16,32 Similarly, our results showed that CIP2A overexpression significantly increased cell proliferation (Figures 2a-d). RNA interference experiments showed that CIP2A silencing leads to a decrease in the proliferation of MM cells, indicating that CIP2A deregulation is an alteration that plays a potential oncogenic role in MM (Figures 2e-h). In addition, CIP2A depletion significantly inhibited the expression of c-Myc and pAkt, two critical substrates of the CIP2A-PP2A axis (Figure 2j). c-Myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation. 10 Given the critical role of c-Myc in promoting cell proliferation, CIP2Amediated c-Myc stabilization may be required for sustained proliferation of MM cells. Our data demonstrated that CIP2A depletion significantly decreased c-Myc expression. Loss of c-Myc is associated with cell cycle transition-controlled proliferation. 33 Thus, our results showed that decreased level of CIP2A correlated with c-Myc, which regulates the proliferation of tumor cells, suggesting that CIP2A plays an important role in the control of MM cell proliferation.
In addition, CIP2A was expressed at higher levels in Dexresistant MM.1R cells, which indicated that CIP2A may participate in Dex therapy. Therefore, we tested the function of CIP2A in Dex therapy. The results showed that the increase of CIP2A expression is associated with Dex resistance and the decrease of CIP2A expression is associated with Dex sensitivity (Figures 3a-d). (h) Tumor tissues were collected from mice; whole-tissue lysates were subjected to western blot using indicated antibodies. (i) Tumor tissues were collected from mice, and PP2A activity was measured by PP2A phosphatase assay. *Po0.05, **P o0.01.
Moreover, CIP2A depletion promoted the effects of Dex-induced apoptosis (Figures 3e and f). Previous studies reported that Akt is a desirable target in MM, a locus and fragility of cancer multidrug resistance. 27,34 We further examined whether the expression of pAkt was altered in the CIP2A-depleted MM.1R cells. As shown in Figure 3g, CIP2A depletion leads to pAkt downregulation and the promotion of Dex-induced apoptosis. Previous studies reported that Akt drives glucocorticoid resistance in acute lymphoblastic leukemia. 13 These results suggest that CIP2A may contribute to Dex resistance through the inhibition of CIP2A-Akt activation.
CIP2A is a cellular PP2A inhibitor, and the CIP2A-PP2A axis is important for CIP2A-induced tumorigenesis. 1 PP2A is a key cellular serine-threonine phosphatase and is an essential tumor suppressor. 35 Our finding that CIP2A is frequently overexpressed in MM is novel and suggests CIP2A deregulation as a possible contributing mechanism to inhibit PP2A. Overexpression of PP2A inhibitors may play an important role in the development of human MM. Therefore, CIP2A inhibition could be a viable strategy to post-translationally target PP2A and inhibit tumor growth in MM. Indeed, CIP2A knockdown significantly increased PP2A activity (Figures 4a and b) and decreased the level of pAkt, as well as the expression of c-Myc (Figure 2j). More importantly, inhibition of PP2A activity reversed these effects (Figures 4c-f). Hence, PP2A inhibition is essential for CIP2Ainduced proliferation.
In vivo, CIP2A depletion in MM cells significantly inhibited the formation of tumors and CIP2A overexpression significantly promoted the formation of tumors in nude mice (Figures 5b-d,  f and g). Moreover, trends of c-Myc, pAkt and PP2Ac expression, and the PP2A activity of CIP2A KD -U266-and U266-transplanted mice were consistent with the results of our in vitro experiments (Figures 5h and i). CIP2A promoted MM proliferation by inhibiting PP2A activity and promoting c-Myc and pAkt expression.
In conclusion, in the present study, we demonstrated that CIP2A is overexpressed and plays an oncogenic role in MM. CIP2A inhibition may provide an important therapeutic strategy for the treatment of MM and Dex-resistant patients by targeting PP2A and facilitating the downregulation of several PP2A-regulated targets, including Akt and c-Myc. On the basis of the data presented here, CIP2A might serve as a molecular target for the development of future MM therapeutics.