Wnt signaling modulator DKK4 inhibits colorectal cancer metastasis through an AKT/Wnt/β-catenin negative feedback pathway

Abstract

Aberrant activation of the Wnt/β-catenin signaling pathway is implicated in most malignant cancers, especially in the initiation and progression of colorectal cancer (CRC). DKK4 is a classical inhibitory molecule of the Wnt/β-catenin pathway, but its role in CRC is ambiguous, and the molecular mechanism remains unclear. Here, we determined DKK4 expression was significantly upregulated in 23 CRC cell lines and 229 CRC tissues when analyzed by quantitative PCR and immunohistochemistry, respectively. Our analysis of tissue samples indicated the survival time of CRC patients with high DKK4 expression was longer than that of patients with medium-low DKK4 expression. We examined the effects of DKK4 on cell proliferation and metastasis by cell counting kit-8 assays, transwell assays, and subcutaneous and metastatic mouse tumor models, and we discovered that DKK4 silencing promoted the metastasis of CRC cells both in vitro and in vivo. Our RNA-seq analysis revealed that AKT2, FZD6, and JUN, which play important roles in AKT and Wnt signaling, were significantly increased after DKK4 knockdown. DKK4 represses Wnt/β-catenin signaling by repressing FZD6 and AKT2/s552 β-catenin in CRC. Further experiments revealed recombinant Wnt3a and LiCl could induce DKK4 expression. Moreover, our bioinformatics analysis and luciferase reporter assays identified posttranscriptional regulators of DKK4 in CRC cells. In summary, DKK4 is elevated in CRC and inhibits cell metastasis by a novel negative feedback mechanism of the Wnt3a/DKK4/AKT/s552 β-catenin regulatory axis to restrict overactivation of Wnt activity in CRC. Therefore, DKK4 restoration may be applied as a potential CRC therapeutic strategy. Aberrant activation of the Wnt/β-catenin signaling pathway is implicated in most malignant cancers, especially in the initiation and progression of colorectal cancer (CRC). DKK4 is a classical inhibitory molecule of the Wnt/β-catenin pathway, but its role in CRC is ambiguous, and the molecular mechanism remains unclear. Here, we determined DKK4 expression was significantly upregulated in 23 CRC cell lines and 229 CRC tissues when analyzed by quantitative PCR and immunohistochemistry, respectively. Our analysis of tissue samples indicated the survival time of CRC patients with high DKK4 expression was longer than that of patients with medium-low DKK4 expression. We examined the effects of DKK4 on cell proliferation and metastasis by cell counting kit-8 assays, transwell assays, and subcutaneous and metastatic mouse tumor models, and we discovered that DKK4 silencing promoted the metastasis of CRC cells both in vitro and in vivo. Our RNA-seq analysis revealed that AKT2, FZD6, and JUN, which play important roles in AKT and Wnt signaling, were significantly increased after DKK4 knockdown. DKK4 represses Wnt/β-catenin signaling by repressing FZD6 and AKT2/s552 β-catenin in CRC. Further experiments revealed recombinant Wnt3a and LiCl could induce DKK4 expression. Moreover, our bioinformatics analysis and luciferase reporter assays identified posttranscriptional regulators of DKK4 in CRC cells. In summary, DKK4 is elevated in CRC and inhibits cell metastasis by a novel negative feedback mechanism of the Wnt3a/DKK4/AKT/s552 β-catenin regulatory axis to restrict overactivation of Wnt activity in CRC. Therefore, DKK4 restoration may be applied as a potential CRC therapeutic strategy. Colorectal cancer (CRC), with more than 1.8 million new cases each year, is the third most frequent cancer globally. More than 861,000 patients die from CRC each year, which is the second leading cause of cancer death (1Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2021; 71: 209-249Google Scholar, 2Arnold M. Sierra M.S. Laversanne M. Soerjomataram I. Jemal A. Bray F. Global patterns and trends in colorectal cancer incidence and mortality.Gut. 2017; 66: 683-691Google Scholar). At the same time, the incidence and mortality of colorectal cancer are also increasing each year in China (3Chen W. Zheng R. Baade P.D. Zhang S. Zeng H. Bray F. et al.Cancer statistics in China.CA Cancer J. Clin. 2016; 66: 115-132Google Scholar, 4Guo P. Huang Z.L. Yu P. Li K. Trends in cancer mortality in China: an update.Ann. Oncol. 2012; 23: 2755-2762Google Scholar). It is well known that early diagnosis and early surgical resection are generally considered to be the most effective ways to reduce CRC mortality. However, as the early symptoms of colorectal cancer are not obvious, an estimated 50% to 60% of CRC patients present with metastases at the time of diagnosis (5Siegel R.L. Miller K.D. Fedewa S.A. Ahnen D.J. Meester R.G.S. Barzi A. et al.Colorectal cancer statistics, 2017.CA Cancer J. Clin. 2017; 67: 177-193Google Scholar). Treatments of advanced CRC include palliative chemotherapy, targeted therapy, and immunotherapy, as well as local treatment. Nevertheless, the 5-years survival rate is only 14% (6Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2019.CA Cancer J. Clin. 2019; 69: 7-34Google Scholar). Therefore, the study of molecular mechanisms and new therapeutic targets for CRC is essential. The Wnt signaling pathway plays an important role in the occurrence and development of colorectal cancer, and the Dickkopf (DKK)family is a classical family of inhibitory molecules in this pathway. Increasing evidence suggests that DKK family members play an important role in the occurrence and development of tumors. The gene family of DKKs encodes the secreted glycoprotein as inhibitory molecules of the typical Wnt signaling pathway, which consists of five members: DKK1-4 and DKKL1 (soggy) (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar). DKK glycoproteins, except soggy, contain an N-terminal signaling peptide and two conserved cysteine-rich domains separated by linkers. The cysteine-rich amino-terminal domain (Cys-1) is unique to each DKK, while the carboxy-terminal cysteine-rich domain (Cys-2) is highly conserved among all DKKs. We previously found that miR-100 and miR-125b promoted Wnt signaling activity by targeting DKK1 and DKK3, leading to cetuximab resistance in CRC (8Lu Y. Zhao X. Liu Q. Li C. Graves-Deal R. Cao Z. et al.lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via wnt/beta-catenin signaling.Nat. Med. 2017; 23: 1331-1341Google Scholar). DKK2 is a functional tumor suppressor that regulates the tumorigenesis of CRC by antagonizing Wnt/β-catenin signaling (9Wang C. Yue Y. Shao B. Qiu Z. Mu J. Tang J. et al.Dickkopf-related protein 2 is epigenetically inactivated and suppresses colorectal cancer growth and tumor metastasis by antagonizing wnt/β-catenin signaling.Cell Physiol. Biochem. 2017; 41: 1709-1724Google Scholar). DKK4 presents inconsistent expression and plays different roles in different cancers. In hepatocellular carcinoma, DKK4 expression is reduced, and its induction suppresses tumor progression and proliferation (10Krupnik V.E. Sharp J.D. Jiang C. Robison K. Chickering T.W. Amaravadi L. et al.Functional and structural diversity of the human Dickkopf gene family.Gene. 1999; 238: 301-313Google Scholar). However, DKK4 is overexpressed in pancreatic cancer, ovarian cancer, gastric cancer, and non-small cell lung cancer. DKK4 activates the MAPK signaling pathway to promote pancreatic cancer progression by regulating VAV3 (11Ouyang Y. Pan J. Tai Q. Ju J. Wang H. Transcriptomic changes associated with DKK4 overexpression in pancreatic cancer cells detected by RNA-seq.Tumour Biol. 2016; 37: 10827-10838Google Scholar) and promotes ovarian cancer metastasis by activating JNK (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar, 12Wang S. Wei H. Zhang S. Dickkopf-4 is frequently overexpressed in epithelial ovarian carcinoma and promotes tumor invasion.BMC Cancer. 2017; 17: 455Google Scholar). In CRC, a few studies with limited samples revealed that DKK4 was upregulated in cancer (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar, 13Lou X. Meng Y. Hou Y. A literature review on function and regulation mechanism of DKK4.J. Cell Mol. Med. 2021; 25: 2786-2794Google Scholar), but little is known about the association of DKK4 expression and CRC prognosis, and the molecular mechanism remains unclear. In the present study, we found that DKK4 is highly expressed among 23 colorectal cancer cell lines and 229 CRC tissues, and high DKK4 expression correlates with a longer survival time of CRC patients. Downregulation of DKK4 promotes the migration and invasion of CRC cells both in vitro and in vivo. To elucidate the specific mechanism, we found that rhWnt3a activated DKK4 expression, and overexpression of DKK4 inhibited Frizzled 6 (FZD6) and AKT2/s552-β-catenin, forming a negative feedback loop that restrained overactivation of Wnt/β-catenin signaling. Moreover, the expression and function of DKK4 was directly inhibited by miR-450b-5p and miR-299-3p. Overall, our studies demonstrated that DKK4 inhibits tumor metastasis in CRC and uncovered a novel regulatory mechanism through the Wnt3a/DKK4/AKT/s552 β-catenin negative feedback loop in CRC. To investigate the DKK4 expression pattern in CRC, we detected DKK4 mRNA levels by quantitative real-time PCR (qRT‒PCR) and protein levels by Western blot in 23 colorectal cancer cell lines and an immortalized human normal intestinal epithelial cell line (FHC). The DKK4 expression level in colorectal cancer cells was significantly higher than that in FHC cells (Fig. 1A). Then, we investigated the expression of DKK4 in the TCGA dataset and found that the expression of DKK4 was significantly increased in CRC tissues compared with normal tissues (Fig. 1B). Immunohistochemistry (IHC) was conducted in three independent CRC tissue microarrays containing 293 cancer lesions. We found that the DKK4 staining intensity was significantly increased in 229 paired primary CRC tissues compared with adjacent normal tissues (Fig. 1, C and D). There were no significant differences in sex, age, tumor location, or clinical stage between the high and medium-low DKK4 expression groups (Table S2). Kaplan‒Meier analysis indicated that the survival of CRC patients in the high DKK4 group was longer than that of patients in the medium-low DKK4 expression group (Fig. 1E). Further analysis showed that the DKK4 high expression group had a longer survival time than the medium-low expression group, especially in pathological grade I/II group compared to that of grade III (Fig. 1F). To further explore the roles of DKK4 in CRC, we established gain-of-function models of DKK4 in Caco-2 and HCT8 cells through lentivirus infection and established loss-of-function models of DKK4 in SW480 and HCT116 cells using a lentiviral vector carrying shRNA (Fig. 2, A and B). Transwell assays showed that DKK4 overexpression decreased the migration and invasion of Caco-2 and HCT8 cells (Fig. 2, C and D). The migration and invasion ability of Caco-2 in DKK4 overexpression group decreased to 51.9% and 31.7% respectively, compared to NC group. And the migration and invasion ability of HCT8 in DKK4 overexpression group decreased to 26.1% and 65.6%, respectively. Conversely, downregulation of DKK4 expression significantly enhanced the migration and invasion of SW480 and HCT116 cells (Fig. 2, E and F). The migration and invasion ability of SW480 in DKK4-downregulated group increased to 2.57 and 1.84 fold respectively, compared to NC group. And the migration and invasion ability of HCT116 in DKK4-downregulated group increased to 2.11 and 2.21 fold, respectively. However, DKK4 did not affect cell proliferation (Fig. 2, G and H). These results indicate that DKK4 inhibits CRC cell migration and invasion but does not influence cell proliferation in vitro. To further validate the effect of DKK4 on metastasis in vivo, HCT116-LV-shDKK4 and control cells were injected into nude mice via the tail vein. After 5 weeks, in vivo bioluminescence imaging showed that the luminescence intensities in the were higher in mice injected with HCT116-LV-shDKK4 cells than in mice in the control group (Fig. The luminescence intensities in the were in mice injected with HCT116-LV-shDKK4 cells than control group. The of metastatic in the was also increased in the HCT116-LV-shDKK4 cell group (Fig. HCT116-LV-shDKK4 and the control cells were injected into nude mice to tumor Downregulation of DKK4 expression showed no on the luminescence growth or tumor between the two groups (Fig. C and D). These results that DKK4 inhibited CRC metastasis in vivo but no effect on To investigate the mechanism by which DKK4 inhibits tumor metastasis in CRC, we RNA-seq to potential a expression as a for expressed we found that were significantly upregulated and were significantly at the mRNA level after DKK4 in HCT116 cells (Fig. The analysis of indicated that the were in and Wnt signaling (Fig. AKT2, FZD6, and JUN, which play important roles in AKT and Wnt signaling Zheng Y. Y. J. H. et of by AKT promotes Biol. Scholar, A. signaling in 2017; Scholar, M. signaling pathway and cell signaling Cancer Scholar), were significantly and Western blot that AKT2, FZD6, and expression levels were decreased after DKK4 overexpression in Caco-2 and HCT8 cells and increased after DKK4 in SW480 and HCT116 cells (Fig. Western blot analysis indicated that overexpression of DKK4 in Caco-2 and HCT8 cells to a in the expression and of and were to β-catenin at J. X. Zhang Q. Z. B. The pathway promotes through its with Cell Scholar). and were also decreased in Caco-2 and HCT8 cells (Fig. In the expression levels of and AKT2, and as well as and the of AKT and Wnt signaling, were significantly increased when DKK4 was in HCT116 and SW480 cells (Fig. However, the expression of and showed no significant between DKK4 upregulated or cells and control cells (Fig. The effect of DKK4 to its in CRC. We examined the cause of DKK4 in CRC cells. DKK4 inhibits Wnt signaling activity and DKK4 to be a of Wnt signaling (10Krupnik V.E. Sharp J.D. Jiang C. Robison K. Chickering T.W. Amaravadi L. et al.Functional and structural diversity of the human Dickkopf gene family.Gene. 1999; 238: 301-313Google Scholar, S. Yu M. Z. I. H. Liu H. et regulates of cells via wnt/β-catenin pathway of 2019; Scholar), we that DKK4 a feedback mechanism of activation of Wnt signaling in the development of CRC. To this we CRC cell lines with recombinant human Wnt3a which is an important of Wnt signaling. We found that with DKK4 mRNA expression was significantly increased at and (Fig. LiCl is the Wnt we cell lines with LiCl and found that the expression of DKK4 was significantly increased at and (Fig. However, no was when cells were with growth (Fig. These that DKK4 overexpression in CRC be a feedback loop of Wnt signaling by growth in the as These results that Wnt3a DKK4 expression and that DKK4 inhibited CRC progression by the of AKT and Wnt signaling. DKK4 expression with survival in CRC patients (Fig. 1E). are important regulators that gene expression at the posttranscriptional We investigated DKK4 was by To this bioinformatics was to potential that The identified that and may to the DKK4 and which were to be in tumorigenesis S. J. Wang Z. Wang K. S. S. as a tumor suppressor via targeting 5 in hepatocellular Scholar, W. Z. Li J. Qiu J. Cao Y. et of promotes of carcinoma by activating axis via Scholar, X. Wang X. X. J. H. Zhao W. regulates cell proliferation and by targeting in Cancer Scholar), in the three by both the and conserved targeting of the three were into SW480 and HCT116 cells. and Western blot that miR-450b-5p and inhibited DKK4 expression (Fig. of miR-450b-5p and were into Caco-2 and HCT8 cells. and Western blot that miR-450b-5p and levels increased DKK4 expression (Fig. To miR-450b-5p and directly to the of we reporter miR-450b-5p and the luciferase activity of the DKK4 reporter this effect was by the of a into the or and C and These results indicate that miR-450b-5p and DKK4 expression by directly targeting its To DKK4 is the functional of miR-450b-5p and we overexpressed miR-450b-5p and in SW480 and HCT116 inhibited levels in Caco-2 and HCT8 and examined effects on migration and Transwell assays revealed that miR-450b-5p or overexpression promoted the migration and invasion of SW480 and HCT116 cells and Conversely, downregulation decreased the migration and invasion of Caco-2 and HCT8 cells and we the and miR-450b-5p or in HCT8 and Caco-2 cells. We found that of miR-450b-5p or into cells the inhibitory effect of DKK4 on migration and invasion and We also found that miR-450b-5p and expression was with DKK4 levels in paired primary CRC tissues and adjacent normal tissues (Fig. These that DKK4 in CRC is by miR-450b-5p and miR-299-3p. In the present study, we found that DKK4 expression was significantly upregulated in primary and metastatic CRC tissues compared with adjacent studies the of DKK4 in CRC cells and tissues (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar), but limited cell lines and tissue Our studies the of DKK4 in CRC by 23 CRC cell lines and tissue microarrays containing 229 CRC cases in DKK4 expression was with of Y. Liu M. S. Li S. M. et clinical of Dickkopf Wnt signaling pathway gene family in and cell Scholar, M. B. C. Zhao L. et of Dickkopf promotes tumor progression via forming the in Med. 2019; Scholar). However, no studies on DKK4 expression in with the of CRC patients. In our study, we found that high DKK4 expression is with survival in colorectal cancer patients. The DKK4 high expression group had a longer survival time than the medium-low expression group, especially in pathological grade I/II group compared to that of grade The of DKK4 plays a significant role in the of initiation and metastasis in cancer demonstrated the role of DKK4 in cell carcinoma, non-small cell lung cancer, and ovarian cancer. In DKK4 is considered a tumor suppressor in cancers as cancer and gastric cancer. These indicate a or function of DKK4 in malignant cancer progression (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar). In CRC, studies revealed that DKK4 increased potential but showed a role in cell migration S. J. X. Li J. DKK4 resistance to and in colorectal cancer 2017; Scholar). Our indicated the tumor role of DKK4 in CRC metastasis and showed that DKK4 had no effect on cell The Wnt/β-catenin signaling pathway is a highly conserved pathway in and its activation is with the malignant progression of human cancers F. Yu C. Li F. Y. Wang Y. L. et signaling in cancers and targeted 2021; Scholar). study indicated that cells with in or β-catenin on Wnt and for a level of β-catenin signaling I. M. et is to high levels of Wnt activity in cancer Scholar). Our are with this and we evidence that DKK4 in CRC cell lines with different DKK family members as DKK1 and DKK2 to with high and expression (7Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene. 2006; 25: 7469-7481Google Scholar), but we did not of expression after DKK4 DKK4 does not function through To explore the further mechanism, the analysis of from RNA-seq indicated that the were in and Wnt signaling and Western blot analysis that FZD6, AKT2, and showed significant changes after DKK4 in SW480 and HCT116 cell molecules as no FZD6 is known as an for Wnt protein in β-catenin signaling which promote CRC progression through the activation of the Wnt/β-catenin pathway C. Jiang C. H. M. L. et promotes colorectal cancer growth and metastasis by the activation of the Wnt/β-catenin pathway via 2019; Scholar). AKT β-catenin at to induce the of β-catenin in the and its activity Zheng Y. Y. J. H. et of by AKT promotes Biol. Scholar, Q. et intestinal cells intestinal Scholar). is a which be in the Wnt signaling pathway and of β-catenin Wang Y. Z.L. Li Li L. and a leading to of Cell Biol. Scholar). We found that DKK4 the Wnt/β-catenin pathway not only by directly FZD6 expression but also by the expression of and leading to the of are of the Wnt/β-catenin and AKT We further that DKK4 could the expression of and is DKK4 increased in the CRC metastasis while plays an inhibitory role on the Wnt/β-catenin We found that rhWnt3a and LiCl could DKK4 expression. Wnt3a is an growth in the CRC tumor that is to this a for DKK4 is upregulated in but plays a our study indicated that rhWnt3a or LiCl DKK4 expression, while overexpressed DKK4 in suppresses Wnt signaling, forming a negative feedback loop to overactivation of the Wnt/β-catenin pathway in CRC. are important regulators that the expression of and function as or tumor suppressor M. in cancer Oncol. 2012; Scholar). To no studies specific that the expression of In this study, we bioinformatics and experiments to the regulatory of DKK4 in CRC cells. and were and demonstrated to and an was between or and DKK4 in CRC The decreased metastatic ability by overexpression of DKK4 was by the expression of miR-450b-5p and miR-299-3p. Overall, our results showed that miR-450b-5p and directly targeted DKK4 in CRC. In summary, we found for the time that DKK4 is upregulated in CRC but plays a role in tumor the mechanism, we identified a novel Wnt3a/DKK4/AKT/s552 β-catenin negative feedback regulatory axis that may Wnt signaling from overactivation in CRC. we found that the expression of DKK4 was by miR-450b-5p and miR-299-3p. These results that DKK4 as a and that the restoration of DKK4 expression may be an effective for the of CRC. The human CRC cell lines and SW480 and the human normal epithelial cell lines FHC and were from the The and of the and HCT116 cell lines are in cells were in with and at in The for cells cetuximab colorectal cancer tissue microarrays and were from the which 229 paired human CRC tissues and the adjacent colorectal tissues and in cases only with primary cancer lesions. primary CRC and adjacent normal tissue samples were from patients CRC at of The samples were in samples were and to be patients and this study was by of staining for DKK4 was on tissue The tissue microarrays were on a for to with at a of and and at with a DKK4 primary The were with and using The were as staining 2 to to 50% to and negative and and intensity of to was as negative or was from cells and CRC tissues with and using a to the was for with a mRNA was with a and the was by real-time PCR by PCR was in using with the The specific to miR-450b-5p and were from The for the of were by and were as for and respectively. The was to the fold changes in the mRNA levels between each and the The PCR were as and CRC cells were in with a and were separated by and The were with in of and with primary and with The were as DKK4 mouse AKT2, FZD6, JUN, and and or was as an The protein were detected with enhanced and by the with DKK4 of and control were and by of and control were and by DKK4 and the negative control were into HCT116 and SW480 and HCT116 and SW480 cells were with and miR-450b-5p and a negative control of the and was using to the carrying the human DKK4 gene was from and targeting DKK4 was from lentiviral vector was as a negative After of CRC cells were with for 2 to cell lines with cells were at a of well in After for and a cell counting kit-8 was to cell proliferation to the The was at using a The cell migration and invasion were by transwell with transwell the migration assays, Caco-2 HCT8 SW480 or negative control cells were in the with an In the invasion of the cells were in the with After at with for an cells that had or through the were with and a to nude mice to 6 were by the of the and in studies were by the mice were into HCT116-LV-shDKK4 and negative control cells were in subcutaneous and tail metastasis in vivo assays, the cells were and in A of 5 cells in of were injected into the of mice The tumor were were at weeks, and the subcutaneous were for in vivo metastasis assays, a of cells in of were injected via the tail into nude mice after the bioluminescence intensity was using the in vivo imaging after the mice were and the lung metastases were for further The indicated cells in were with miR-450b-5p or or negative control and the indicated or using were and for luciferase assays after A was to the luciferase The luciferase activity was to luciferase activity and as the luciferase assays were in These and HCT116 cell lines were for human rhWnt3a or LiCl or was in SW480 cell and cells at and were and with Then, was to The Cancer were to the expression of DKK4 mRNA in human cancer and normal from at three independent experiments were analyzed using and are as the and and the were for between two respectively. were analyzed using of were compared using the were to among clinical was considered of a significant and The analyzed in the present study are from the on The that no of with the of this The present study was by from the of China and and the of X. and X. Z. J. L. W. and L. J. L. and W. L. J. and X. Z. and X. Z. and X. Z. and X. Z. with and 2

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