piRNA-independent function of PIWIL1 as a co-activator for anaphase promoting complex/cyclosome to drive pancreatic cancer metastasis

Abstract

Piwi proteins are normally restricted in germ cells to suppress transposons through associations with Piwi-interacting RNAs (piRNAs), but they are also frequently activated in many types of human cancers. A great puzzle is the lack of significant induction of corresponding piRNAs in cancer cells, as we document here in human pancreatic ductal adenocarcinomas (PDACs), which implies that such germline-specific proteins are somehow hijacked to promote tumorigenesis through a different mode of action. Here, we show that in the absence of piRNAs, human PIWIL1 in PDAC functions as an oncoprotein by activating the anaphase promoting complex/cyclosome (APC/C) E3 complex, which then targets a critical cell adhesion-related protein, Pinin, to enhance PDAC metastasis. This is in contrast to piRNA-dependent PIWIL1 ubiquitination and removal by APC/C during late spermiogenesis. These findings unveil a piRNA-dependent mechanism to switch PIWIL1 from a substrate in spermatids to a co-activator of APC/C in human cancer cells.

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Fig. 1: PIWIL1 is largely present in the apo state, without piRNA binding, in human cancer cells.
Fig. 2: PIWIL1 acts as a novel co-activator of the APC/C E3 complex.
Fig. 3: PIWIL1 leads to the constitutive activation of APC/C in PDAC cells.
Fig. 4: The APC/C co-activator function of PIWIL1 is required for its role in PDAC metastasis but not tumour growth.
Fig. 5: Pinin is a substrate of APC/CPIWIL1 in PDAC cells.
Fig. 6: Pinin inhibits cell migration and metastasis but not cell proliferation and tumour growth in PDAC cells.
Fig. 7: Ectopic Pinin overrides the stimulatory effect of PIWIL1 on PDAC metastasis, but not tumour growth.
Fig. 8: Comparison of PIWIL1 and Pinin expression in human PDAC specimens.

Data availability

The RNA sequencing data supporting the findings of this study have been deposited in the GEO repository under accession code GSE141340. The MS data have been deposited in ProteomeXchange with the primary accession code PXD017452. The human cancer data were derived from the TCGA Research Network at http://cancergenome.nih.gov/. The dataset derived from this resource that supports the findings of this study can be downloaded from BROAD (http://firebrowse.org/viewGene.html?gene=piwil1). All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Change history

  • 15 April 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank X.-D. Fu from the University of California, San Diego, Y. Zheng from UT Southwestern and members of the M.-F.L. Lab for helpful comments. We thank L. Zhan from the Institute of Nutrition and Health, SIBS, CAS, Y. Yu from Tianjin Medical University, and Q. Shi from the University of Science and Technology of China for experimental assistance. This work was supported by grants from the National Key R&D Program of China (2017YFA0504400), the National Natural Science Foundation of China (91940305, 31830109, 31821004, 31961133022 and 91640201), the Chinese Academy of Sciences (“Strategic Priority Research Program” grant XDB19010203) and the Shanghai Municipal Science and Technology Major Project (17JC1420100, 19JC1410200 and 2017SHZDZX01).

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Contributions

M.-F.L. and S.Z. planned the project. F.L., P.Y., M.R., Y.L., L.W., T.L., W.L., J.L., D.L., Y.L., Y.-F.R., S.Z. and M.-F.L. designed the experiments. F.L., P.Y., M.R., W.T., C.-H.J., Y.-P.H., F.Z., T.W., Q.Y. and S.Z. conducted the experiments. F.L., P.Y., S.Z. and M.-F.L. analysed the data. F.L., P.Y., S.Z., D.L. and M.-F.L. wrote the paper. All authors discussed the results and commented on the manuscript. M.-F.L. and S.Z. supervised the overall work.

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Correspondence to Shuang Zhao or Mo-Fang Liu.

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Extended data

Extended Data Fig. 1 Piwil1 (Hiwi) is aberrantly expressed in various types of human tumors and cancer cell lines.

a, Comparison of Piwil1 expression between primary tumor (red column) and paired adjacent normal tissues (blue column) in various types of human cancers from TCGA database, with the rectangle indicating the range between the third and the first quartile values and the segment inside the rectangle indicating the median values, the whiskers indicating the maximum (top) and minimum (bottom) values, and the unfilled circles as outlier values indicating variations in each box-whisker plot dataset. BLCA, Bladder urothelial carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, Cholangiocarcinoma; COAD, Colon adenocarcinoma; COADREAD, Colorectal adenocarcinoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; LGG, Lower grade glioma; HNSC, Head and neck squamous cell carcinoma; KICH, Kidney chromophobe; KICH, Pan-kidney cohort; KIPAN, KICH+KIRC+KIRP; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PCPG, Pheochromocytoma and paraganglioma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; STAD, Stomach adenocarcinoma; STES, Stomach and esophageal carcinoma; THCA, Thyroid carcinoma; THYM, Thymoma; UCEC, Uterine corpus endometrial carcinoma. The data were downloaded from BROAD (http://firebrowse.org/viewGene.html?gene=piwil1). b, c, qRT-PCR (b) and western blot analyses (c) of Piwil1 expression in various types of human cancer cell lines (left) and PDAC cell lines (right), with 293 T (lane 1) and Flag-PIWIL1-transfected 293 T cells (lane 2) serving as negative and positive controls, respectively. Results shown in b are mean ± SD of three separate experiments. Statistical analysis was performed using two-tailed Student t test. Results shown in c are representative of three independent experiments. Unprocessed blots and statistical source data are provided in Source Data Extended Data Fig. 1 and Statistical Source Data Extended Data Fig. 1, respectively. Source data

Extended Data Fig. 2 Piwil1 knockdown inhibits the proliferation and migration of PDAC cells.

a-h, Piwil1 knockdown in BxPC-3 cells reduced cell proliferation (a, n=4 independent experiments), anchorage-independent growth (b, n=3 independent experiments), xenograft tumor growth in nude mice (c, n=3 animals), cell migration (d, n=3 independent experiments), invasion (e, n=3 independent experiments), and metastatic colonization in NOD/SCID mice through either orthotopic (f, n=3 animals), spleen (g, n=3 animals) or tail vein injection (h, n=3 animals). il, Piwil1 knockdown in AsPC-1 cells reduced cell proliferation (i, n=4 independent experiments), anchorage-independent growth (j, n=3 independent experiments), cell migration (k, n=3 independent experiments) and invasion (l, n=3 independent experiments). BxPC-3 and AsPC-1 cells were respectively transfected with Piwil1 siRNA (a, b, d, e and i–l) or infected with shPiwil1 pseudovirus (c and f–h), and the assays were performed at 24 h post-transfection. a and i, Cell proliferation by MTT assay. b and j, Soft agar colony formation assay, with the relative number of soft agar foci per field. c, Xenograft assay in nude mice, with the time course of xenograft tumour growth. d and k, Wound healing assay, with the quantitative results of wound closure. e and l, Transwell invasion assay, with the invasive cell numbers per field at 24 h after the cells plated. f, Orthotopic xenograft assay in NOD/SCID mice. Left, the average volumes of xenograft tumors in the pancreata; right, quantification of liver metastases area. g and h, Liver or lung metastatic colonization assays in NOD/SCID mice through spleen (g) or tail vein injection (h), respectively, with the bioluminescence quantification of metastasis tumors. The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Ectopic expression of Piwil1 promotes the proliferation and migration of PDAC cells.

Ectopic expression of Piwil1 in PANC-1 cells potently promoted cell proliferation (a, n=4 independent experiments), anchorage-independent growth (b, n=3 independent experiments), xenograft tumor growth in nude mice (c, n=3 animals), cell migration (d, n=3 independent experiments), invasion (e, n=3 independent experiments), and metastatic colonization in NOD/SCID mice through either orthotopic (f, n=3 animals), spleen (g, n=3 animals) or tail vein injection (h, n=3 animals). PANC-1 cells were respectively transfected with Flag-PIWIL1 or control vector p3×Flag, and the assays were performed at 24 h post-transfection. The procedures are similar to that described in Extended Data Fig. 2. The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Cyclin B1 is not a direct substrate for APC/CPIWIL1 E3 complex in PDAC cells.

a, PIWIL1 was persistently associated with APC/C complex in AsPC-1 cells during the cell cycle. Both HeLa (lanes 1–4) and AsPC-1 cells (lanes 5–8) were synchronized in G1/S phase, then released and harvested at indicated time points for anti-APC3 co-IP assay of the association of Cdc20, Cdh1, or PIWIL1 with APC/C (top), and cell cycles were examined by FACS (bottom). The phosphorylated APC3 was indicated by “*”. b, c, Piwil1 knockdown (c) or overexpression (d) barely affected Cyclin B1 ubiquitination in PDAC cells. BxPC-3 and AsPC-1 cells were co-transfected with pEF-HA-Ub and scramble siRNA (lanes 1, 4, 7 and 10), Cdc20 siRNA (lanes 2, 3, 8 and 9) or Piwil1 siRNAs (lanes 5, 6, 11 and 12), respectively. PANC-1, Capan-1 and MIA PaCa-2 cells were co-transfected with pEF-HA-Ub and p3xFlag control vector (lanes 1, 3, 5, 7, 9 and 11) or p3xFlag-PIWIL1 (lanes 2, 4, 6, 8, 10 and 12), respectively. Top, anti-Cyclin B1 IP pellets were immunoblotted by anti-Ub for Cyclin B1-Ub conjugates (upper) or anti-Cyclin B1 to normalize the loading amounts (lower). Bottom, cell lysates were immunoblotted for PIWIL1, Cdc20, and Cyclin B1, with β-actin serving as a loading control. d, PIWIL1 depletion did not alter the expression pattern of Cyclins and progression of cell cycle in BxPC-3 cells. 24 h post-transfection with Piwil1 siRNA (lanes 7–12) or control scramble siRNA (lanes 1–6), BxPC-3 cells were synchronized in G1/S phase, then released and harvested at indicated time points for immunoblotting of indicated proteins in cell lysates (left), or FACS analyses of cell cycles (right). e, Gating strategy for analysis of synchronized cells. Results shown in ad are representative of three independent experiments. Unprocessed blots are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 The APC/C co-activator function is required for PIWIL1 action on cell migration but not proliferation in PDAC cells.

ad, C-box mutations in PIWIL1 did not alter its effect on cell proliferation (a, n=4 independent experiments), anchorage-independent growth (b, n=3 independent experiments), but markedly impaired its stimulatory effect on cell migration (c, n = 3 independent experiments) and invasion (d, n=3 independent experiments) in MIA PaCa-2 cells. The cells were respectively transfected with control p3×Flag or Flag-tagged wildtype PIWIL1, PIWIL1-C-boxmut, PIWIL1-N or PIWIL1-N-C-boxmut, and the assays were performed at 24 h post-transfection. eh, The co-activator-deficient C-box mutant MIWI effectively resorted cell proliferation (e, n=5 independent experiments) and anchorage-independent growth (f, n= 3 independent experiments), but failed to rescue cell migration (g, n=3 independent experiments) and invasion (h, n=3 independent experiments) in PIWIL1-depleted BxPC-3 cells. The cells were respectively co-transfected with Piwil1 siRNAs and control vector p3×Flag or Flag-tagged MIWI or MIWI-C-boxmut, and the assays were performed at 24 h post-transfection. The procedures are similar to that described in Extended Data Fig. 2. The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 Downregulation of IDH3B, ACTL6A or ARPC3 by PIWIL1 is not involved in its oncogenic function in PDAC cells.

a–d, Ectopic expression of IDH3B, ACTL6A or ARPC3 barely affected cell proliferation (a, n=4 independent experiments), anchorage-independent growth (b, n=3 independent experiments), cell migration (c, n=3 independent experiments) and invasion (d, n=3 independent experiments) in BxPC-3 cells. The cells were respectively transfected with Myc-tagged IDH3B, ACTL6A, ARPC3 or control vector pCMV-Myc, and the assays were performed at 24 h post-transfection. e–h, Co-transfection of Myc-tagged IDH3B, ACTL6A or ARPC3 vectors barely affected the stimulatory effect of PIWIL1 on cell proliferation (e, n=4 independent experiments), anchorage-independent growth (f, n=3: independent experiments), cell migration (c, n=3 independent experiments) and invasion (d, n=3 independent experiments) in PANC-1 cells. The cells were respectively co-transfected with Flag-PIWIL1 and Myc-tagged IDH3B, ACTL6A, or ARPC3, with pCMV-Myc and Myc-Pinin respectively serving as negative and positive controls, and the assays were performed at 24 h post-transfection. The procedures are similar to that described in Extended Data Fig. 2. The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 Pinin is specifically ubiquitinated by APC/CPIWIL1.

a, Cdh1 or Cdc20 knockdown marginally altered Pinin ubiquitination in BxPC-3 cells. Top, anti-Pinin IP pellets were immunoblotted with anti-Ub or anti-Pinin; bottom, cell lysates were immunoblotted for indicated proteins, with β-actin serving as a loading control. The cells were transfected with indicated siRNAs (lanes 1–4), with Apc10 siRNAs (lane 5) serving as positive control. b, The PIWIL1-N peptide, but not its C-box mutant, promoted Pinin ubiquitination by APC/C in vitro. Left, in vitro Pinin ubiquitination using PIWIL1-N peptides (lanes 6 and 7), with omission of one component or more (lanes 1–5) as negative controls; right, Coomassie blue staining of recombinant PIWIL1-N peptides. PIWIL1-N peptides and Pinin were bacterially expressed and purified. c, The D-box (red) and KEN-box (green) are conserved in vertebrate Pinin. d–g, Double mutations of D-Box and KEN-box resulted in Pinin resistant to ubiquitination by APC/CPIWIL1. (d), Schematic representation of the construction of D-Box or KEN-box mutant Pinin. (e), Pinin ubiquitination in PIWIL1 co-transfected-PANC-1 cells. Myc-Pinin or its mutants was respectively co-transfected with Flag-PIWIL1 and HA-Ub. Top, anti-Myc IP pellets were immunoblotted with anti-Ub or anti-Myc; bottom, cell lysates were immunoblotted for Flag-PIWIL1 or Myc-Pinin, with β-actin serving as a loading control. (f), Ubiquitination of Pinin or Pinin-DK mutant by APC/C in vitro. (g), The stability of Pinin proteins in PIWIL1-expressing PANC-1 cells. Myc-tagged Pinin (lanes 1–6) or Pinin-DK mutant (lanes 7–12) was co-transfected with Flag-PIWIL1. 48 h post-transfection, cells were treated with 50 μg/ml cycloheximide and harvested for immunoblotting of PIWIL1 or Pinin at indicated time points, with β-actin serving as a loading control. Results shown in a, b and eg are representative of three independent experiments. Unprocessed blots are provided in Source Data Extended Data Fig. 7. Source data

Extended Data Fig. 8 Pinin inhibits cell migration and metastasis but not cell proliferation and tumor growth in PDAC cells.

Pinin knockdown in PANC-1 cells barely altered cell proliferation (a, n=3 independent experiments), anchorage-independent growth (b, n=3 independent experiments), and xenograft tumor growth in nude mice (c, n=3 animals), but promoted cell migration (d, n=3 independent experiments), invasion (e, n=3 independent experiments), and metastatic colonization in NOD/SCID mice through either orthotopic (f, n=3 animals), spleen (g, n=3 animals) or tail vein injection (h, n=3 animals). The cells were respectively transfected with Pinin siRNA (Pinin-siR1 or Pinin-siR2) or scrambled siRNA (scr siR) (a, b, d, and e), or shPinin or control pSilencer (c and fh), and the assays were performed at 24 h post-transfection. The procedures are similar to that described in Extended Data Fig. 2. The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 8. Source data

Extended Data Fig. 9 Pinin knockdown rescues the inhibitory effect of PIWIL1 depletion on PDAC metastasis but not tumor growth.

a–d, Co-transfection of Myc-Pinin in MIA PaCa-2 cells barely affected the stimulatory effect of PIWIL1 on cell proliferation (a, n=4 independent experiments) and anchorage-independent growth (b, n=3 independent experiments), but effectively attenuated the stimulatory effect of PIWIL1 on cell migration (c, n=3 independent experiments) and invasion (d, n=3 independent experiments). The cells were co-transfected with indicated vectors, and the assays were performed at 24 h post-transfection. eh, Pinin knockdown in BxPC-1 cells barely affected the inhibitory effect of PIWIL1 depletion on cell proliferation (e, n=6 independent experiments) and anchorage-independent growth (f, n=3 independent experiments), but effectively attenuated the inhibitory effect of PIWIL1 depletion on cell migration (g, n=3 independent experiments) and invasion (h, n=3 independent experiments) in cultured cells and metastatic colonization in the liver of NOD/SCID mice through either orthotopic or spleen (i, j, n=3 animals). The cells were co-transfected with indicated siRNAs (eh) or shRNAs (i and j), and the assays were performed at 24 h post-transfection. The procedures are similar to that described in Extended Data Fig. 2. k, Dispase-based dissociation assay showed the functional requirement of APC/CPIWIL1-Pinin axis in controlling intercellular cohesion in BxPC-3 cells. The cells were transfected with indicated siRNAs, and the assay was performed at 48 h post-transfection. Left, representative images; right, the average fragment number from 6 independent experiments, reporting as Tukey box-plots (middle lines, median values; lower and upper sides of the rectangles, the 1st and 3rd percentile, whiskers, confidence interval). The mean ± SD was plotted in all graphs. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 9. Source data

Extended Data Fig. 10 Clinical relevance of the APCPIWIL1-Pinin axis in PDAC patients.

a, Comparison of PIWIL1 staining intensity between early and late stages of acinar-to-ductal metaplasia (ADM). Representative images of Hematoxylin and eosin (H&E), α-Amylase, CK19 or PIWIL1 immunohistochemical staining (brown) of pancreas sections of chronic pancreatitis specimens without ADM (top), or with ADM at early (middle) or late stages (bottom), respectively. Magnification for pancreas sections, 40×; scale bars, 100 µm. b, Kaplan–Meier curves for overall survival analysis of the 30 patients recruited to our study with PIWIL1 (top) or Pinin expression (bottom), respectively. c, PIWIL1 and Pinin immunohistochemical (IHC) staining of serial sections from human primary PDAC tumor (middle panels), paired adjacent normal tissue (top panels) and liver metastatic nodule (bottom panels) from the 7 patients that showed liver metastasis after surgery. Scale bar, 100 μm. Bottom panels in each row showing an enlargement of black framed region on the top. d, Comparison of PIWIL1 (left) and Pinin IHC staining intensity (right) between primary PDAC tumor and paired liver metastatic nodule sections from the 7 patients. The positively stained cells were analyzed and the average values of staining intensity were calculated using Image-Pro Plus software, and then normalized with those in paired adjacent normal tissues. Results shown in ad are representative of three independent experiments. Statistical analysis was performed using two-tailed Student t test. Statistical source data are provided in Statistical Source Data Extended Data Fig. 10. Source data

Supplementary information

Reporting Summary

Supplementary Tables

Table 1: The list of proteins upregulated in PIWIL1 siRNA-transfected BxPC-3 cells relative to Scr siR control. Table 2: Relevance of PIWIL1 and Pinin expression with clinicopathological features in PDAC. Table 3: The sequences of chemically synthesized DNA and RNA oligonucleotides. Table 4: The list of antibodies and reagents.

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Li, F., Yuan, P., Rao, M. et al. piRNA-independent function of PIWIL1 as a co-activator for anaphase promoting complex/cyclosome to drive pancreatic cancer metastasis. Nat Cell Biol 22, 425–438 (2020). https://doi.org/10.1038/s41556-020-0486-z

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