b94fffb9bf15739006853dfc38ae4ff7 pmc_34884726.pdf 7f8955b6e55270de4a5e627cff9f38b6c82475fa pmc_34884726.pdf 3122ffdb4872e5e96b0571847cd6e48648e8ef94912389be5fa122f97d07f36c pmc_34884726.pdf Title: Dickkopf-1 Inhibition Reactivates Wnt/-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo Subject: The Wnt/-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to -catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression. Keywords: Wnt antagonists; Dickkopf proteins; DKK; -catenin; Wnt pathway; rhabdomyosarcoma; differentiation Author: Irina Giralt, Gabriel Gallo-Oller, Natalia Navarro, Patricia Zarzosa, Guillem Pons, Ainara Magdaleno, Miguel F. Segura, Constantino Sábado, Raquel Hladun, Diego Arango, José Sánchez de Toledo, Lucas Moreno, Soledad Gallego and Josep Roma Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Nov 30 02:09:40 2021 CET ModDate: Tue Nov 30 06:43:10 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3379784 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 HHFREY+EURM7 Type 1 Builtin yes yes no 15 0 PSSUWS+VnURWPalladioL Type 1 Custom yes yes no 19 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 23 0 JFMNPS+URWPalladioL-Ital Type 1 Custom yes yes no 27 0 EWCDYM+URWPalladioL-Bold Type 1 Custom yes yes no 32 0 UGDIYG+URWPalladioL-Roma Type 1 Custom yes yes no 36 0 ZYYJXI+EURB10 Type 1 Builtin yes yes no 40 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 48 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 73 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 77 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 81 0 LBBPFO+PazoMath-Italic Type 1 Builtin yes yes no 105 0 TELXLI+CMSY10 Type 1 Builtin yes yes no 130 0 UZKIAN+CMEX10 Type 1 Builtin yes yes no 137 0 GIGFZE+CMR10 Type 1 Builtin yes yes no 141 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:30:10 CET RepresentationInformation: pmc_34884726.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-11 05:06:49 CET Size: 3379784 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 343 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Analysis of DKK Expression in RMS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Genetic DKK-1 Inhibition Led to Reduced Proliferation in RMS Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: DKK-1 Knockdown Activated Wnt Signaling and Induced Expression of Myogenic Markers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: DKK-1 Pharmacological Inhibition Decreased Cell Proliferation and Invasion Rates Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: WAY-262611 Treatment Produced an Increase in -Catenin Levels, an Induction of Myogenic Markers and a Decrease in FAK Levels Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: WAY-262611 Treatment Decreased the Survival of Intravenously Injected Tumor Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Database and mRNA Expression Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Lentiviral Transduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Cell Proliferation Assay and IC50 Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Transwell Cell Invasion Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Luciferase Assay of -Catenin Transcriptional Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Primary Tumor and Metastasis Models in Mice Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: Dickkopf-1 Inhibition Reactivates Wnt/-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo Author: Irina Giralt, Gabriel Gallo-Oller, Natalia Navarro, Patricia Zarzosa, Guillem Pons, Ainara Magdaleno, Miguel F. Segura, Constantino Sábado, Raquel Hladun, Diego Arango, José Sánchez de Toledo, Lucas Moreno, Soledad Gallego and Josep Roma Subject: The Wnt/-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to -catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression. Keywords: Wnt antagonists; Dickkopf proteins; DKK; -catenin; Wnt pathway; rhabdomyosarcoma; differentiation Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Nov 30 02:09:40 CET 2021 ModDate: Tue Nov 30 06:43:10 CET 2021 ID: 0x553c5d79a1a1fb3e471c8d859f65912b, 0x553c5d79a1a1fb3e471c8d859f65912b Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: EWCDYM+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: EWCDYM+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TELXLI+CMSY10 FontSubset: true FirstChar: 2 LastChar: 48 FontDescriptor: FontName: TELXLI+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: PSSUWS+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: PSSUWS+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UGDIYG+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: UGDIYG+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: ZYYJXI+EURB10 FontSubset: true FirstChar: 12 LastChar: 12 FontDescriptor: FontName: ZYYJXI+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: LBBPFO+PazoMath-Italic FontSubset: true FirstChar: 98 LastChar: 112 FontDescriptor: FontName: LBBPFO+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: UZKIAN+CMEX10 FontSubset: true FirstChar: 16 LastChar: 17 FontDescriptor: FontName: UZKIAN+CMEX10 Flags: Symbolic FontBBox: -24, -2960, 1454, 772 FontFile: true Font: BaseFont: GIFXFT+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 178 FontDescriptor: FontName: GIFXFT+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JFMNPS+URWPalladioL-Ital FontSubset: true FirstChar: 44 LastChar: 121 FontDescriptor: FontName: JFMNPS+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: GIGFZE+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: GIGFZE+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: HHFREY+EURM7 FontSubset: true FirstChar: 12 LastChar: 12 FontDescriptor: FontName: HHFREY+EURM7 Flags: Symbolic FontBBox: -5, -243, 1229, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 181, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 100, 642, 111, 654 Annotation: Subtype: Link Rect: 217, 642, 229, 654 Annotation: Subtype: Link Rect: 395, 642, 406, 654 Annotation: Subtype: Link Rect: 479, 642, 490, 654 Annotation: Subtype: Link Rect: 214, 628, 225, 642 Annotation: Subtype: Link Rect: 327, 628, 338, 642 Annotation: Subtype: Link Rect: 230, 616, 241, 629 Annotation: Subtype: Link Rect: 340, 616, 351, 629 Annotation: Subtype: Link Rect: 132, 359, 154, 367 Annotation: Subtype: Link Rect: 35, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 81, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 411, 181, 418, 189 ActionDest: 149 Annotation: Subtype: Link Rect: 462, 93, 469, 102 ActionDest: 149 Annotation: Subtype: Link Rect: 469, 92, 476, 102 ActionDest: 149 Annotation: Subtype: Link Rect: 375, 68, 382, 76 ActionDest: 149 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 420, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 482, 87, 501 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 305, 737, 312, 746 ActionDest: 149 Annotation: Subtype: Link Rect: 315, 737, 322, 746 ActionDest: 149 Annotation: Subtype: Link Rect: 482, 686, 489, 695 ActionDest: 149 Annotation: Subtype: Link Rect: 363, 649, 370, 658 ActionDest: 149 Annotation: Subtype: Link Rect: 261, 611, 268, 620 ActionDest: 149 Annotation: Subtype: Link Rect: 347, 611, 353, 620 ActionDest: 149 Annotation: Subtype: Link Rect: 443, 611, 450, 620 ActionDest: 149 Annotation: Subtype: Link Rect: 260, 586, 267, 595 ActionDest: 149 Annotation: Subtype: Link Rect: 416, 573, 428, 582 ActionDest: 149 Annotation: Subtype: Link Rect: 485, 536, 497, 545 ActionDest: 149 Annotation: Subtype: Link Rect: 363, 498, 375, 507 ActionDest: 149 Annotation: Subtype: Link Rect: 258, 473, 269, 482 ActionDest: 149 Annotation: Subtype: Link Rect: 240, 306, 247, 318 ActionDest: 93 Annotation: Subtype: Link Rect: 200, 243, 207, 255 ActionDest: 93 Annotation: Subtype: Link Rect: 220, 246, 232, 255 ActionDest: 149 Annotation: Subtype: Link Rect: 303, 218, 310, 230 ActionDest: 93 Annotation: Subtype: Link Rect: 240, 117, 247, 129 ActionDest: 93 Annotation: Subtype: Link Rect: 397, 105, 404, 117 ActionDest: 93 Page: Sequence: 3 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 398, 666, 405, 678 ActionDest: 93 Annotation: Subtype: Link Rect: 488, 641, 495, 653 ActionDest: 93 Annotation: Subtype: Link Rect: 410, 604, 417, 616 ActionDest: 93 Annotation: Subtype: Link Rect: 529, 591, 536, 603 ActionDest: 93 Annotation: Subtype: Link Rect: 486, 566, 493, 578 ActionDest: 93 Annotation: Subtype: Link Rect: 257, 503, 264, 515 ActionDest: 109 Annotation: Subtype: Link Rect: 542, 465, 550, 477 ActionDest: 109 Annotation: Subtype: Link Rect: 389, 440, 396, 452 ActionDest: 109 Annotation: Subtype: Link Rect: 343, 428, 350, 440 ActionDest: 109 Annotation: Subtype: Link Rect: 535, 367, 542, 379 ActionDest: 109 Annotation: Subtype: Link Rect: 435, 316, 442, 328 ActionDest: 109 Annotation: Subtype: Link Rect: 424, 304, 431, 316 ActionDest: 109 Annotation: Subtype: Link Rect: 246, 216, 253, 228 ActionDest: 115 Annotation: Subtype: Link Rect: 289, 153, 296, 165 ActionDest: 115 Annotation: Subtype: Link Rect: 389, 115, 396, 127 ActionDest: 115 Annotation: Subtype: Link Rect: 541, 90, 548, 102 ActionDest: 115 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 233, 732, 240, 744 ActionDest: 115 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 358, 147, 365, 156 ActionDest: 149 Annotation: Subtype: Link Rect: 541, 134, 548, 143 ActionDest: 149 Annotation: Subtype: Link Rect: 549, 134, 556, 143 ActionDest: 149 Annotation: Subtype: Link Rect: 375, 109, 381, 118 ActionDest: 149 Annotation: Subtype: Link Rect: 364, 97, 371, 106 ActionDest: 149 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 280, 697, 292, 706 ActionDest: 149 Annotation: Subtype: Link Rect: 292, 697, 304, 706 ActionDest: 149 Annotation: Subtype: Link Rect: 315, 660, 327, 669 ActionDest: 149 Annotation: Subtype: Link Rect: 330, 659, 342, 669 ActionDest: 149 Annotation: Subtype: Link Rect: 327, 634, 339, 643 ActionDest: 152 Annotation: Subtype: Link Rect: 524, 622, 536, 631 ActionDest: 152 Annotation: Subtype: Link Rect: 229, 609, 241, 618 ActionDest: 152 Annotation: Subtype: Link Rect: 313, 546, 325, 556 ActionDest: 152 Annotation: Subtype: Link Rect: 325, 547, 337, 556 ActionDest: 152 Annotation: Subtype: Link Rect: 338, 496, 350, 505 ActionDest: 152 Annotation: Subtype: Link Rect: 272, 459, 278, 468 ActionDest: 149 Annotation: Subtype: Link Rect: 279, 459, 291, 468 ActionDest: 152 Annotation: Subtype: Link Rect: 309, 383, 316, 392 ActionDest: 149 Annotation: Subtype: Link Rect: 238, 333, 249, 342 ActionDest: 152 Annotation: Subtype: Link Rect: 250, 333, 262, 342 ActionDest: 152 Annotation: Subtype: Link Rect: 236, 220, 243, 229 ActionDest: 149 Annotation: Subtype: Link Rect: 440, 208, 452, 217 ActionDest: 152 Annotation: Subtype: Link Rect: 453, 208, 465, 217 ActionDest: 152 Annotation: Subtype: Link Rect: 280, 170, 292, 179 ActionDest: 152 Annotation: Subtype: Link Rect: 191, 132, 203, 141 ActionDest: 152 Annotation: Subtype: Link Rect: 203, 132, 215, 141 ActionDest: 152 Annotation: Subtype: Link Rect: 239, 120, 250, 129 ActionDest: 152 Annotation: Subtype: Link Rect: 314, 107, 326, 116 ActionDest: 152 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 346, 685, 358, 694 ActionDest: 152 Annotation: Subtype: Link Rect: 320, 660, 332, 669 ActionDest: 152 Annotation: Subtype: Link Rect: 271, 609, 283, 618 ActionDest: 152 Annotation: Subtype: Link Rect: 224, 509, 236, 518 ActionDest: 152 Annotation: Subtype: Link Rect: 232, 496, 244, 505 ActionDest: 152 Annotation: Subtype: Link Rect: 261, 233, 273, 242 ActionDest: 152 Annotation: Subtype: Link Rect: 274, 233, 286, 242 ActionDest: 152 Annotation: Subtype: Link Rect: 416, 233, 428, 242 ActionDest: 152 Annotation: Subtype: Link Rect: 241, 182, 253, 192 ActionDest: 152 Annotation: Subtype: Link Rect: 444, 103, 518, 115 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 205, 682, 407, 694 Annotation: Subtype: Link Rect: 286, 672, 298, 681 ActionDest: 149 Annotation: Subtype: Link Rect: 210, 657, 341, 669 Annotation: Subtype: Link Rect: 410, 471, 422, 480 ActionDest: 152 Annotation: Subtype: Link Rect: 473, 169, 485, 178 ActionDest: 152 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 392, 84, 404, 93 ActionDest: 152 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 419, 391, 560, 402 Annotation: Subtype: Link Rect: 166, 379, 264, 390 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 113, 541, 151, 551 Annotation: Subtype: Link Rect: 313, 517, 350, 528 Annotation: Subtype: Link Rect: 357, 517, 393, 528 Annotation: Subtype: Link Rect: 510, 506, 547, 517 Annotation: Subtype: Link Rect: 335, 471, 373, 482 Annotation: Subtype: Link Rect: 257, 438, 295, 448 Annotation: Subtype: Link Rect: 301, 438, 337, 448 Annotation: Subtype: Link Rect: 440, 414, 478, 425 Annotation: Subtype: Link Rect: 395, 391, 433, 402 Annotation: Subtype: Link Rect: 439, 391, 475, 402 Annotation: Subtype: Link Rect: 455, 368, 492, 379 Annotation: Subtype: Link Rect: 59, 334, 97, 344 Annotation: Subtype: Link Rect: 104, 311, 142, 321 Annotation: Subtype: Link Rect: 148, 311, 184, 321 Annotation: Subtype: Link Rect: 241, 287, 278, 298 Annotation: Subtype: Link Rect: 520, 264, 557, 275 Annotation: Subtype: Link Rect: 239, 241, 276, 252 Annotation: Subtype: Link Rect: 283, 241, 319, 252 Annotation: Subtype: Link Rect: 512, 207, 550, 218 Annotation: Subtype: Link Rect: 464, 184, 502, 195 Annotation: Subtype: Link Rect: 519, 161, 557, 172 Annotation: Subtype: Link Rect: 59, 150, 95, 160 Annotation: Subtype: Link Rect: 258, 126, 295, 137 Annotation: Subtype: Link Rect: 375, 103, 413, 114 Annotation: Subtype: Link Rect: 419, 103, 455, 114 Annotation: Subtype: Link Rect: 109, 70, 146, 80 Annotation: Subtype: Link Rect: 153, 70, 189, 80 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 438, 709, 476, 720 Annotation: Subtype: Link Rect: 482, 709, 518, 720 Annotation: Subtype: Link Rect: 354, 676, 391, 686 Annotation: Subtype: Link Rect: 397, 676, 433, 686 Annotation: Subtype: Link Rect: 131, 641, 169, 651 Annotation: Subtype: Link Rect: 59, 607, 97, 617 Annotation: Subtype: Link Rect: 103, 607, 139, 617 Annotation: Subtype: Link Rect: 398, 583, 436, 594 Annotation: Subtype: Link Rect: 235, 560, 272, 571 Annotation: Subtype: Link Rect: 440, 537, 478, 548 Annotation: Subtype: Link Rect: 484, 537, 520, 548 Annotation: Subtype: Link Rect: 489, 525, 526, 536 Annotation: Subtype: Link Rect: 113, 503, 151, 513 Annotation: Subtype: Link Rect: 161, 480, 199, 490 Annotation: Subtype: Link Rect: 205, 480, 241, 490 Annotation: Subtype: Link Rect: 154, 446, 191, 456 Annotation: Subtype: Link Rect: 283, 422, 321, 433 Annotation: Subtype: Link Rect: 327, 422, 363, 433 Annotation: Subtype: Link Rect: 467, 399, 504, 410 Annotation: Subtype: Link Rect: 178, 364, 216, 375 Annotation: Subtype: Link Rect: 95, 342, 133, 352 Annotation: Subtype: Link Rect: 139, 342, 175, 352 Annotation: Subtype: Link Rect: 173, 319, 210, 329 Annotation: Subtype: Link Rect: 331, 295, 369, 306 Annotation: Subtype: Link Rect: 375, 295, 411, 306 Annotation: Subtype: Link Rect: 453, 272, 491, 283 Annotation: Subtype: Link Rect: 442, 249, 479, 260 Annotation: Subtype: Link Rect: 486, 249, 522, 260 Annotation: Subtype: Link Rect: 118, 215, 155, 226 Annotation: Subtype: Link Rect: 146, 181, 184, 191 Annotation: Subtype: Link Rect: 190, 181, 226, 191 Checksum: 55592b9a Type: CRC32 Checksum: b94fffb9bf15739006853dfc38ae4ff7 Type: MD5 Checksum: 7f8955b6e55270de4a5e627cff9f38b6c82475fa Type: SHA-1