b784effda5811e14164cdc666a2e557e pmc_10342163.pdf cc881cb783310358a6e6396484c502cb5abb99d9 pmc_10342163.pdf 94861f299e70331e637350cf8b005cc7006daf33f671b9706bac6bccd652063f pmc_10342163.pdf Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells Subject: We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E. Keywords: thyroid cancer (TC); low-density lipoprotein receptor (LDLR); BRAF V600E; RAS/RAF/MAPK (MEK)/ERK pathway; low-density lipoprotein (LDL) Author: Giovanna Revilla, Lara Ruiz-Auladell, Núria Fucui Vallverdú, Paula Santamaría, Antonio Moral, José Ignacio Pérez, Changda Li, Victoria Fuste, Enrique Lerma, Rosa Corcoy, Fabián Pitoia, Joan Carles Escolà-Gil and Eugènia Mato Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jul 12 11:58:17 2023 CEST ModDate: Wed Jul 12 12:02:14 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3566787 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SNDHFT+VnURWPalladioL Type 1 Custom yes yes no 10 0 NCDZTB+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 MNNSLW+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 IPFWJJ+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 XEYJZU+CMSY10 Type 1 Builtin yes yes no 28 0 CKNIOL+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 50 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 75 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 87 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 91 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 95 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 99 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 105 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 111 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 126 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 130 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 134 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 138 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 144 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 150 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 161 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 165 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 169 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 173 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 179 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 185 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 201 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 205 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 209 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 213 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 219 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 225 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 241 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 245 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 249 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 253 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 259 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 265 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 283 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 287 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 291 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 295 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 301 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 316 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 320 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 324 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 328 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 334 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 340 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 361 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 365 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 369 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 373 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 379 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 385 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-05-04 02:42:10 CEST RepresentationInformation: pmc_10342163.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-04-24 02:40:36 CEST Size: 3566787 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 626 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Differential Regulation of Low-Density Lipoprotein Receptor (LDLR) Protein Expression and Low-Density Lipoprotein (LDL) Uptake in Papillary Thyroid Carcinoma (PTC) Cell Lines with Different Mutational Status after LDL Incubation Destination: subsection.2.1 Item: Title: LDL Promotes Higher Proliferation Levels and Decreases Adhesion Capacity in the BCPAP Cell Line, in Comparison to the Constant Proliferation and Cellular Migration Promoted by LDL in the TPC1 Cell Line Destination: subsection.2.2 Item: Title: LDL Enhances the RAS/RAF/MAPK (MEK)/ERK Pathway Destination: subsection.2.3 Item: Title: Vemurafenib Reduces Cellular Proliferation Promoted by LDL, Downregulating LDLR Expression and Impairing RAS/RAF/MAPK (MEK)/ERK Pathway Activation in BCPAP Cell Line Destination: subsection.2.4 Item: Title: LDLR Is Required for LDL-Mediated Induction of RAS/RAF/MAPK (MEK)/ERK Pathway, LDL-Uptake and Cell Proliferation in the BCPAP Cell Line Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Lines and Cell Culture Destination: subsection.4.1 Item: Title: Human LDL Isolation Destination: subsection.4.2 Item: Title: Labeling LDL with DiI Destination: subsection.4.3 Item: Title: Analysis of Lipoprotein Uptake by a Fluorometric Assay Destination: subsection.4.4 Item: Title: Analysis of Lipoprotein Uptake by Confocal Microscopy Destination: subsection.4.5 Item: Title: Quantitative Real-Time PCR Destination: subsection.4.6 Item: Title: Protein Extraction and Western Blot Destination: subsection.4.7 Item: Title: MTT Assay Destination: subsection.4.8 Item: Title: Migration and Adhesion Assay Destination: subsection.4.9 Item: Title: Transient Transfection Assay Destination: subsection.4.10 Item: Title: Statistical Analysis Destination: subsection.4.11 Item: Title: References Destination: section.A. Info: Title: Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells Author: Giovanna Revilla, Lara Ruiz-Auladell, Núria Fucui Vallverdú, Paula Santamaría, Antonio Moral, José Ignacio Pérez, Changda Li, Victoria Fuste, Enrique Lerma, Rosa Corcoy, Fabián Pitoia, Joan Carles Escolà-Gil and Eugènia Mato Subject: We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E. Keywords: thyroid cancer (TC); low-density lipoprotein receptor (LDLR); BRAF V600E; RAS/RAF/MAPK (MEK)/ERK pathway; low-density lipoprotein (LDL) Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jul 12 11:58:17 CEST 2023 ModDate: Wed Jul 12 12:02:14 CEST 2023 ID: 0x1e5c1b4c86513ba892a5ccbe06dd78c8, 0x1e5c1b4c86513ba892a5ccbe06dd78c8 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: CKNIOL+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: CKNIOL+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MNNSLW+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: MNNSLW+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IPFWJJ+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: IPFWJJ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SNDHFT+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: SNDHFT+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: XEYJZU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: XEYJZU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: NCDZTB+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: NCDZTB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 86, 293, 152, 302 Annotation: Subtype: Link Rect: 34, 281, 107, 290 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 128, 662, 139, 674 Annotation: Subtype: Link Rect: 195, 648, 206, 662 Annotation: Subtype: Link Rect: 446, 648, 457, 662 Annotation: Subtype: Link Rect: 162, 636, 173, 649 Annotation: Subtype: Link Rect: 143, 17, 286, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 390, 87, 409 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 267, 723, 274, 732 ActionDest: 402 Annotation: Subtype: Link Rect: 241, 685, 248, 694 ActionDest: 402 Annotation: Subtype: Link Rect: 397, 660, 404, 669 ActionDest: 405 Annotation: Subtype: Link Rect: 407, 660, 414, 669 ActionDest: 405 Annotation: Subtype: Link Rect: 391, 622, 398, 631 ActionDest: 405 Annotation: Subtype: Link Rect: 228, 560, 235, 569 ActionDest: 405 Annotation: Subtype: Link Rect: 236, 560, 243, 569 ActionDest: 405 Annotation: Subtype: Link Rect: 523, 535, 530, 544 ActionDest: 405 Annotation: Subtype: Link Rect: 530, 535, 542, 544 ActionDest: 405 Annotation: Subtype: Link Rect: 460, 509, 472, 519 ActionDest: 405 Annotation: Subtype: Link Rect: 508, 484, 519, 493 ActionDest: 405 Annotation: Subtype: Link Rect: 522, 484, 534, 493 ActionDest: 405 Annotation: Subtype: Link Rect: 502, 447, 514, 456 ActionDest: 405 Annotation: Subtype: Link Rect: 468, 422, 480, 431 ActionDest: 405 Annotation: Subtype: Link Rect: 451, 384, 458, 393 ActionDest: 405 Annotation: Subtype: Link Rect: 458, 384, 470, 393 ActionDest: 405 Annotation: Subtype: Link Rect: 544, 371, 556, 380 ActionDest: 405 Annotation: Subtype: Link Rect: 350, 346, 362, 355 ActionDest: 405 Annotation: Subtype: Link Rect: 362, 346, 374, 355 ActionDest: 405 Annotation: Subtype: Link Rect: 278, 309, 290, 318 ActionDest: 405 Annotation: Subtype: Link Rect: 508, 271, 515, 280 ActionDest: 405 Annotation: Subtype: Link Rect: 515, 271, 527, 280 ActionDest: 405 Annotation: Subtype: Link Rect: 528, 271, 540, 280 ActionDest: 405 Annotation: Subtype: Link Rect: 472, 133, 479, 145 ActionDest: 79 Annotation: Subtype: Link Rect: 261, 58, 268, 70 ActionDest: 79 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 262, 695, 269, 707 ActionDest: 79 Annotation: Subtype: Link Rect: 295, 695, 302, 707 ActionDest: 118 Annotation: Subtype: Link Rect: 199, 204, 206, 216 ActionDest: 118 Annotation: Subtype: Link Rect: 541, 178, 548, 190 ActionDest: 118 Annotation: Subtype: Link Rect: 206, 82, 213, 94 ActionDest: 118 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 240, 695, 247, 707 ActionDest: 79 Annotation: Subtype: Link Rect: 444, 72, 451, 84 ActionDest: 193 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 544, 733, 550, 745 ActionDest: 193 Annotation: Subtype: Link Rect: 266, 695, 273, 707 ActionDest: 193 Annotation: Subtype: Link Rect: 394, 96, 401, 108 ActionDest: 233 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 374, 708, 381, 720 ActionDest: 233 Annotation: Subtype: Link Rect: 271, 115, 278, 127 ActionDest: 275 Annotation: Subtype: Link Rect: 455, 78, 462, 90 ActionDest: 79 Annotation: Subtype: Link Rect: 489, 78, 495, 90 ActionDest: 233 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 410, 720, 417, 732 ActionDest: 275 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 376, 626, 383, 638 ActionDest: 275 Annotation: Subtype: Link Rect: 195, 614, 202, 626 ActionDest: 118 Annotation: Subtype: Link Rect: 475, 601, 482, 613 ActionDest: 275 Annotation: Subtype: Link Rect: 432, 490, 439, 502 ActionDest: 308 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 330, 497, 337, 509 ActionDest: 308 Annotation: Subtype: Link Rect: 381, 447, 388, 459 ActionDest: 308 Annotation: Subtype: Link Rect: 335, 384, 342, 396 ActionDest: 308 Annotation: Subtype: Link Rect: 478, 372, 485, 384 ActionDest: 308 Annotation: Subtype: Link Rect: 228, 311, 240, 320 ActionDest: 405 Annotation: Subtype: Link Rect: 243, 311, 255, 320 ActionDest: 405 Annotation: Subtype: Link Rect: 400, 286, 412, 295 ActionDest: 405 Annotation: Subtype: Link Rect: 393, 236, 405, 245 ActionDest: 405 Annotation: Subtype: Link Rect: 405, 236, 417, 245 ActionDest: 405 Annotation: Subtype: Link Rect: 420, 236, 432, 245 ActionDest: 408 Annotation: Subtype: Link Rect: 264, 148, 276, 157 ActionDest: 405 Annotation: Subtype: Link Rect: 242, 110, 253, 120 ActionDest: 408 Annotation: Subtype: Link Rect: 256, 110, 268, 120 ActionDest: 408 Annotation: Subtype: Link Rect: 268, 73, 280, 82 ActionDest: 408 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 410, 723, 422, 732 ActionDest: 408 Annotation: Subtype: Link Rect: 423, 723, 434, 732 ActionDest: 408 Annotation: Subtype: Link Rect: 385, 698, 397, 707 ActionDest: 408 Annotation: Subtype: Link Rect: 189, 447, 201, 456 ActionDest: 408 Annotation: Subtype: Link Rect: 504, 396, 516, 406 ActionDest: 408 Annotation: Subtype: Link Rect: 519, 397, 531, 406 ActionDest: 408 Annotation: Subtype: Link Rect: 365, 321, 377, 330 ActionDest: 408 Annotation: Subtype: Link Rect: 220, 258, 232, 267 ActionDest: 408 Annotation: Subtype: Link Rect: 233, 258, 245, 267 ActionDest: 408 Annotation: Subtype: Link Rect: 435, 246, 447, 255 ActionDest: 408 Annotation: Subtype: Link Rect: 391, 158, 403, 167 ActionDest: 408 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 368, 698, 380, 707 ActionDest: 408 Annotation: Subtype: Link Rect: 381, 698, 393, 707 ActionDest: 408 Annotation: Subtype: Link Rect: 419, 622, 431, 631 ActionDest: 408 Annotation: Subtype: Link Rect: 373, 472, 380, 481 ActionDest: 405 Annotation: Subtype: Link Rect: 380, 472, 392, 481 ActionDest: 408 Annotation: Subtype: Link Rect: 395, 472, 407, 481 ActionDest: 411 Annotation: Subtype: Link Rect: 443, 406, 450, 418 ActionDest: 353 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 168, 630, 367, 642 Annotation: Subtype: Link Rect: 343, 442, 354, 451 ActionDest: 411 Annotation: Subtype: Link Rect: 355, 442, 367, 451 ActionDest: 411 Annotation: Subtype: Link Rect: 530, 291, 542, 300 ActionDest: 411 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 213, 434, 225, 443 ActionDest: 411 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 189, 509, 201, 519 ActionDest: 411 Annotation: Subtype: Link Rect: 202, 509, 214, 519 ActionDest: 411 Annotation: Subtype: Link Rect: 453, 270, 465, 279 ActionDest: 411 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 486, 622, 498, 631 ActionDest: 411 Annotation: Subtype: Link Rect: 499, 622, 511, 631 ActionDest: 411 Annotation: Subtype: Link Rect: 513, 597, 527, 607 ActionDest: 393 Annotation: Subtype: Link Rect: 545, 597, 559, 607 ActionDest: 396 Annotation: Subtype: Link Rect: 479, 420, 560, 431 Annotation: Subtype: Link Rect: 165, 408, 324, 419 Annotation: Subtype: Link Rect: 187, 120, 225, 130 Annotation: Subtype: Link Rect: 231, 120, 267, 130 Annotation: Subtype: Link Rect: 140, 85, 177, 95 Annotation: Subtype: Link Rect: 184, 85, 220, 95 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 59, 711, 96, 721 Annotation: Subtype: Link Rect: 245, 688, 283, 698 Annotation: Subtype: Link Rect: 324, 664, 362, 675 Annotation: Subtype: Link Rect: 131, 642, 168, 652 Annotation: Subtype: Link Rect: 438, 618, 476, 629 Annotation: Subtype: Link Rect: 482, 618, 518, 629 Annotation: Subtype: Link Rect: 59, 584, 96, 594 Annotation: Subtype: Link Rect: 455, 560, 493, 571 Annotation: Subtype: Link Rect: 432, 537, 470, 550 Annotation: Subtype: Link Rect: 104, 504, 141, 514 Annotation: Subtype: Link Rect: 95, 481, 132, 491 Annotation: Subtype: Link Rect: 138, 481, 174, 491 Annotation: Subtype: Link Rect: 59, 458, 96, 468 Annotation: Subtype: Link Rect: 102, 458, 138, 468 Annotation: Subtype: Link Rect: 131, 435, 168, 445 Annotation: Subtype: Link Rect: 175, 435, 211, 445 Annotation: Subtype: Link Rect: 313, 411, 351, 422 Annotation: Subtype: Link Rect: 127, 389, 164, 399 Annotation: Subtype: Link Rect: 95, 354, 133, 364 Annotation: Subtype: Link Rect: 238, 319, 275, 330 Annotation: Subtype: Link Rect: 282, 319, 318, 330 Annotation: Subtype: Link Rect: 59, 262, 96, 272 Annotation: Subtype: Link Rect: 241, 238, 279, 249 Annotation: Subtype: Link Rect: 320, 205, 358, 215 Annotation: Subtype: Link Rect: 364, 205, 400, 215 Annotation: Subtype: Link Rect: 222, 181, 260, 192 Annotation: Subtype: Link Rect: 410, 158, 448, 169 Annotation: Subtype: Link Rect: 454, 158, 490, 169 Annotation: Subtype: Link Rect: 288, 135, 326, 146 Annotation: Subtype: Link Rect: 59, 113, 96, 123 Annotation: Subtype: Link Rect: 411, 100, 449, 111 Annotation: Subtype: Link Rect: 455, 100, 491, 111 Annotation: Subtype: Link Rect: 515, 77, 553, 88 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 118, 711, 155, 721 Annotation: Subtype: Link Rect: 397, 699, 434, 710 Annotation: Subtype: Link Rect: 59, 665, 96, 675 Annotation: Subtype: Link Rect: 392, 641, 429, 652 Annotation: Subtype: Link Rect: 351, 618, 389, 629 Annotation: Subtype: Link Rect: 395, 618, 431, 629 Annotation: Subtype: Link Rect: 140, 596, 177, 606 Annotation: Subtype: Link Rect: 184, 596, 220, 606 Annotation: Subtype: Link Rect: 303, 560, 341, 571 Annotation: Subtype: Link Rect: 347, 560, 383, 571 Annotation: Subtype: Link Rect: 448, 537, 486, 548 Annotation: Subtype: Link Rect: 442, 514, 480, 525 Annotation: Subtype: Link Rect: 377, 491, 414, 502 Annotation: Subtype: Link Rect: 136, 469, 174, 479 Annotation: Subtype: Link Rect: 320, 445, 357, 456 Annotation: Subtype: Link Rect: 144, 423, 182, 433 Annotation: Subtype: Link Rect: 188, 423, 224, 433 Annotation: Subtype: Link Rect: 219, 388, 257, 399 Annotation: Subtype: Link Rect: 263, 388, 299, 399 Annotation: Subtype: Link Rect: 519, 365, 557, 376 Annotation: Subtype: Link Rect: 59, 354, 95, 364 Annotation: Subtype: Link Rect: 319, 319, 357, 330 Annotation: Subtype: Link Rect: 95, 297, 133, 307 Annotation: Subtype: Link Rect: 86, 262, 123, 272 Annotation: Subtype: Link Rect: 237, 239, 275, 249 Annotation: Subtype: Link Rect: 326, 215, 364, 226 Annotation: Subtype: Link Rect: 407, 192, 444, 203 Annotation: Subtype: Link Rect: 265, 170, 302, 180 Annotation: Subtype: Link Rect: 378, 146, 415, 157 Annotation: Subtype: Link Rect: 190, 113, 227, 123 Annotation: Subtype: Link Rect: 233, 113, 269, 123 Annotation: Subtype: Link Rect: 142, 78, 179, 88 Annotation: Subtype: Link Rect: 186, 78, 222, 88 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 144, 722, 182, 733 Annotation: Subtype: Link Rect: 296, 688, 334, 698 Annotation: Subtype: Link Rect: 367, 664, 405, 675 Annotation: Subtype: Link Rect: 411, 664, 447, 675 Annotation: Subtype: Link Rect: 140, 642, 177, 652 Annotation: Subtype: Link Rect: 183, 642, 219, 652 Annotation: Subtype: Link Rect: 451, 618, 489, 629 Annotation: Subtype: Link Rect: 495, 606, 533, 617 Annotation: Subtype: Link Rect: 212, 572, 249, 583 Annotation: Subtype: Link Rect: 338, 537, 376, 548 Annotation: Subtype: Link Rect: 237, 503, 275, 514 Annotation: Subtype: Link Rect: 281, 503, 317, 514 Checksum: 11159891 Type: CRC32 Checksum: b784effda5811e14164cdc666a2e557e Type: MD5 Checksum: cc881cb783310358a6e6396484c502cb5abb99d9 Type: SHA-1 Checksum: 94861f299e70331e637350cf8b005cc7006daf33f671b9706bac6bccd652063f Type: SHA-256