Apatinib weakens proliferation, migration, invasion, and angiogenesis of thyroid cancer cells through downregulating pyruvate kinase M2

Thyroid cancer (TC) is the most frequent malignancy of the endocrine system. Apatinib, as an anti-angiogenic agent, has been applied in the therapy of several cancers. However, the function and mechanism of Apatinib in TC have not been clearly elucidated. After processing with Apatinib alone or combined PKM2 overexpression plasmids, cell proliferation, migration, and invasion were analyzed by EdU staining, CCK-8, wound healing, and Transwell. Meanwhile. HUVECs were incubated with the conditioned medium prepared from cell culture medium, and tube formation and VEGFR2 expression in HUVECs were examined using tube formation and immunofluorescence (IF) assays. Besides, we established a nude mouse xenograft model by lentivirus-mediated PKM2 shRNAs, and tested the growth of tumors; the pathological structure was analyzed with H&E staining. And the expressions of N-cadherin, Vimentin, E-cadherin, PKM2, VEGFA, VEGFR2, and Ki67 were determined by immunohistochemistry or Western blot. Apatinib could prominently suppress proliferation, migration, invasion, and HUVEC tube formation in SW579 and TPC-1 cells. Besides, we discovered that Apatinib had a significant inhibitory role on the expression of pyruvate kinase M2 (PKM2) in TC cells. And PKM2 overexpression also could notably reverse Apatinib-mediated inhibition of TC progression. Moreover, PKM2 shRNAs were applied to TC xenografts, resulting in significant reduction in tumor volume and suppression of angiogenesis-related protein expression. In summary, Apatinib has a regulatory role in TC progression, and Apatinib can block cancer cell angiogenesis by downregulating PKM2. This will provide a theoretical basis for therapy of TC.

In line with the above study, we further verified the roles of PKM2 in the malignant progression of TC mediated by Apatinib.We overexpressed PKM2 in Apatinib-treated SW579 and TPC-1 cells.EdU staining data exhibited that the inhibitory effect of Apatinib on the proliferative activity of SW579 and TPC-1 cells could be prominently reversed by PKM2 overexpression (Fig. 3A).Subsequently, the CCK8 assay further verified that the overexpression of PKM2 significantly enhanced the Apatinib-reduced cell proliferation observed in SW579 and TPC-1 cells (Fig. 3B).Wound healing results indicated that PKM2 overexpression observably attenuated the blocking role of Apatinib on the migration of SW579 and TPC-1 cells (Fig. 3C).And Transwell data also represented that PKM2 overexpression also could markedly enhance the invasion capacity of SW579 and TPC-1 cells, which was suppressed by Apatinib (Fig. 3D).Furthermore, overexpression of PKM2 triggered EMT that is inhibited by Apatinib, via enhancement of N-cadherin, Vimentin and inhibition of E-cadherin in SW579 and TPC-1 cells (Fig. 3E).Thus, we demonstrated that Apatinib can prevent TC process by downregulating PKM2.

PKM2 overexpression dramatically attenuated the inhibitory effect of Apatinib on HUVEC tube formation in SW579 and TPC-1 cells
Additionally, we also studied the influence of PKM2 overexpression on HUVEC tube formation.HUVECs were incubated with the tumor-conditioned medium of SW579 and TPC-1 cells, which have been processed with Apatinib and PKM2 overexpression plasmid.The results from tube formation assay signified that Apatinib memorably reduced the angiogenesis capacity of HUVECs, which also could be notably restored by PKM2 overexpression in SW579 and TPC-1 cells (Fig. 4A).Meanwhile, the data of IF staining represented that addition of Apatinib in SW579 and TPC-1 cells signally reduced the expression of VEGFR2 in HUVECs, which also could be dramatically weakened by PKM2 overexpression (Fig. 4B).Similarly, IF result also indicated that Apatinib could prominently downregulate VEGFA by PKM2(Fig.4C).In addition, western blot data manifested that PKM2 overexpression could prominently attenuate the downregulation of PKM2 and VEGFA expressions mediated by Apatinib (Fig. 4D).Overall, this part of the data confirmed that Apatinib can prevent angiogenesis of HUVECs by downregulating PKM2.As proved by the in vitro results, PKM2 overexpression could induce proliferation, metastasis and HUVEC tube formation of Apatinib-treated SW579 and TPC-1 cells.We further confirmed the impacts of PKM2 silencing on the growth, pathologic structure, and angiogenesis of TC grafted tumor in nude mice.As denoted in Fig. 5A,B, relative to the control group, the tumor was markedly reduced in the PKM2 silencing group.Then H&E staining results displayed that in the control group, thyroid tumor cells of nude mice had normal and clear outlines, abundant cytoplasm, obvious nucleoli and heterotypy; in the PKM2-silenced group, thyroid tumor cells were seen to have necrotic cells, increased nuclei division and blurred cell outlines; the number of necrotic thyroid cells in the PKM2-silenced group was reduced compared with that in the control group (Fig. 5C).Besides, IHC data indicated that knockdown of PKM2 could lower PKM2, VEGFA, VEGFR2, and Ki67 expressions in the grafted tumors (Fig. 5D).Simultaneously, western blot results also showed that PKM2 knockdown could lead to a remarkable downregulation of N-cadherin, Vimentin, PKM2, and VEGFA expression in tumor tissues, while E-cadherin expression was notably upregulated (Fig. 5E,F).So, these data testified that PKM2 knockdown had a significant attenuating effect on the development of TC in vivo.

Discussion
TC has a high incidence and its causative mechanisms have not been specifically elucidated clinically 4 .On account of the literatures, the causes of TC are relevant to radiation, genetics, poor lifestyle habits, and abnormal iodine intake 26 .Currently, most TC have slow progression and can achieve good prognosis and long survival time after standard treatment 27 .However, some patients still have the risk of tumor recurrence, metastasis or www.nature.com/scientificreports/death.Meanwhile, advanced TC is more difficult to treat and has a poor prognosis 28 .Therefore, it is extremely key to take timely and effective measures for the therapy of patients with advanced TC.
A key feature of tumor growth and metastasis is aberrant angiogenesis, in which the vascular endothelial growth factor (VEGF) pathway plays a key role 29 .Research proved that tumor vascular endothelial cells can secrete VEGF, which can participate in the generation of new microvessels and cause an imbalance of endothelial growth and inhibition 30 .The formation of nascent microvessels can also create a hypoxic environment, which induces VEGF transcription and exacerbates tumor microangiogenesis 31 .VEGF receptor (VEGFR) is the key receptor in VEGF pathway 32 .Overexpression of VEGF-A, VEGFR1 and VEGFR2 has been observed in more than 90% of TC patients 33,34 .VEGFR activation can accelerate endothelial cell proliferation, survival, migration and invasion, thereby increasing vascular permeability to induce tumor angiogenesis 35 .Research proved that high expression of VEGF and VEGFR was associated with tumor growth, metastasis, microvessel density, and poor patient prognosis in TC 36 .Suppression of tumor angiogenesis has become a novel strategy for targeted tumor therapy.Currently, anti-angiogenic drugs have made significant advances in TC therapy.However, there are multiple disadvantages of anti-angiogenic drug, such as expensive, unstable efficacy, large adverse effects, and unknown mechanisms of drug resistance, etc.Therefore, anti-angiogenic targeted therapies in TC still need to be studied in depth.
Apatinib, as anti-angiogenic targeted drug, is vital for the clinical therapy of malignant tumors 37 .Research demonstrated that Apatinib can selectively compete with VEGFR2 binding sites in cells to prevent angiogenesis in tumor tissues 38 .Multiple researches also demonstrated the potential function of Apatinib in cancer progression 39,40 .In recent years, Apatinib has also been reported in TC.For instance, researches suggested that Apatinib can induce autophagy and apoptosis in human TC cells 16 ; Apatinib has an inhibitory effect on angiogenesis of TC cells 41 .In our study, we further testified that Apatinib could obviously suppress proliferation, migration, invasion, as well as HUVEC tube formation of TC cells.and Apatinib also could downregulate VEGFR2 and VEGFA in TC.Thus, we further confirmed the noteworthy blocking effect of Apatinib on the malignant process of TC.
Tumor cells adopted aerobic glycolysis as the main mode of energy supply 18,42 .Pyruvate kinase (PK), a key enzyme in the glycolysis, can catalyze the production of pyruvate from phosphoenolpyruvate 43 .Among them, PKM2 is one of the isozymes of PK and displays overexpression in multiple of tumor cells 21,44 , and exists primarily as an enzymatically inactive monomer or dimer.Some evidences indicated that nuclear PKM2 could promote the Warburg effect and cell cycle progression in cancer cells and contributes to tumorigenesis 45 .Research certified that tumor cells can acquire the metabolic property of preferential glycolysis by expressing PKM2, which also enables them to obtain vast energy to maintain their high proliferation and metastasis 21 .Besides, PKM2 can also affect the growth of tumor cells by regulating the products and of cellular metabolic processes and related cytokines 46 .However, there are no reports about PKM2 in TC.In our study, we data also Apatinib memorably downregulated PKM2 in TC cells.Moreover, we proved that PKM2 is required for Apatinib-mediated inhibition of TC cell proliferation, metastasis, and angiogenesis.And we disclosed that PKM2 knockdown could prevent subcutaneous tumor growth and angiogenesis in vivo.The PKM2 dimer is capable of migrating into the nucleus, where interacts directly with the HIF-1α and regulate expression of numerous pro-glycolytic enzymes 47 .Lactate, as an end product of glycolysis, stimulates angiogenesis by increasing the production of VEGF and VEGFR2 in HUVECs 48 .Thus, this could elucidate how Apatinib leads to decreased expression of VEGF by inhibiting PKM2 and consequently inhibits angiogenesis.
Lin et al. 49 have reported in their phase II clinical trial that Apatinib has shown therapeutic efficacy in radioiodine-refractory differentiated thyroid cancer (RAIR-DTC), achieving an objective response rate (ORR) of 80% and a disease control rate (DCR) of 95%.The study by Du et al. 50has also found that the patients undergoing Apatinib for RAIR-DTC, have achieved an ORR of 80% and a DCR of 90%.Zhang et al. 14 have reported a case of an inoperable locally advanced DTC patient, who has undergone a curative operation after the treatment of preoperative monotherapy of apatinib.Currently, most TC-related studies have focused on the clinical efficacy observation and safety analysis of Apatinib and its combination with other drug therapy, while the mechanism of anti-cancer effect of Apatinib has been less studied.Our study is the first to demonstrate that Apatinib can achieve anti-TC effects by altering PKM2 expression in tumor cells.This might provide a new idea for the antitumor role of Apatinib.

Cell culture
SW579 and TPC-1 cells were purchased from the ATCC.HUVECs were purchased from ScienCell (USA).SW579 cells were incubated in L-15 medium (GIBCO, Cat.no.41300039), and TPC-1 cells grown in Minimum essential medium (MEM; GIBCO, Cat.no.41500034).HUVECs were cultured in ECM medium (Life Technologies).The cultures all contained 10% fetal bovine serum (Gibco, USA).And both types of cells were all incubated in an incubator at 37 °C with 5% CO 2 .

Cell treatment
SW579 and TPC-1 cells were first disposed of 20 ng/ml, 50 ng/ml, 100 ng/ml Apatinib (Selleck Chemicals; cat.no.S5248) for 48 h 51 .PKM2 overexpression plasmid and empty vector, PKM2 shRNAs and control were provided by Integrated Biotech Solutions (Shanghai, China).SW579 and TPC-1 cells were (density about 60%) in 6-well plate were transfected with PKM2 overexpression plasmid and vector by applying Lipofectamine 3000 (Invitrogen) in accordance with the reagent instructions.PKM2 shRNAs lentivirus was packaged by WZ Bioscience (Shandong, China).And lentivirus was applied to infect SW579 cells at MOI = 50, and then added with 5 mg/ml Polybrene (Santa Cruz).After 96 h of infection, cells were added with 4 μg/ml Puromycin to screen stable expression cells.

Figure 1 .
Figure 1.Apatinib markedly restrained proliferation migration, and invasion of SW579 and TPC-1 cells.(A) CCK8 was used to measure the cell proliferation of SW579 and TPC-1 cells.*P < 0.05, **P < 0.01, vs. Blank.(B) EdU staining was used to detect cell proliferation of of SW579 and TPC-1 cells.(C) Wound healing demonstrated the change of cell migration ability in Apatinib-treated SW579 and TPC-1 cells.Magnification, 100 ×. (D) Cell invasion was evaluated through Transwell assay in SW579 and TPC-1 cells after processing with Apatinib.(E) Western blot was utilized to analyze the change of N-cadherin, Vimentin, E-cadherin expressions in SW579 and TPC-1 cells after administration with Apatinib.Magnification, 200 ×.

Figure 2 .Figure 3 .
Figure 2. Apatinib prominently suppressed HUVEC tube formation and weakened PKM2 expression in TC. (A) Images of tube formation assay in HUVECs, which were incubated with the conditioned medium from Apatinib-treated SW579 and TPC-1 cells.Magnification, 100 ×. (B) After incubation with the conditioned medium, IF staining was conducted to assess the expression change of VEGFR2 in HUVECs.Magnification, 200 ×.(C) The expression change of VEGFA was credited through IF staining in HUVECs after treatment with the conditioned medium from Apatinib-processed SW579 and TPC-1 cells.Magnification, 200 ×.(D) Western blot was utilized to analyze the change of VEGFA and PKM2 expressions in SW579 and TPC-1 cells after administration with Apatinib.

Figure 4 .
Figure 4. PKM2 overexpression dramatically attenuated the inhibitory effect of Apatinib on HUVEC tube formation in SW579 and TPC-1 cells.(A) Apatinib-processed SW579 and TPC-1 cells were then transfected with PKM2 overexpression plasmid.After incubation with the conditioned medium from the treated SW579 and TPC-1 cells, the angiogenesis was assessed by applying tube formation assay in HUVECs.Magnification, 100 ×. (B) The change in VEGFR2 expression was examined through IF staining in the processed HUVECs.Magnification, 200 ×.(C) IF staining was adopted to evaluate the change of VEGFA expression in each group.Magnification, 200 ×.(D) Western blot displayed the change levels of PKM2 and VEGFA expressions in SW579 and TPC-1 cells.

Figure 5 .
Figure 5. PKM2 silencing prevented subcutaneous tumor growth and angiogenesis in a nude mouse TC model SW579 cells stably transfected with PKM2 shRNAs were subcutaneously injected into nude mice.(A) Subcutaneous tumor cells were excised and displayed.(B) Tumor volume was tested and calculated every 5 days for 30 days.(C) H&E staining presented the change in pathological structure.Magnification, 200 ×.(D) IHC assay was applied to certify the changes in PKM2, VEGFA, VEGFR2, and Ki67 expressions in each group of tumors.Magnification, 200 ×.(E) The expression changes of N-cadherin, Vimentin, E-cadherin were tested using western blot in tumors.(F) The expression changes of PKM2 and VEGFA were tested using western blot in tumors.