An RGDKGE-Containing Cryptic Collagen Fragment Regulates Phosphorylation of Large Tumor Suppressor Kinase-1 and Controls Ovarian Tumor Growth by a Yes-Associated Protein–Dependent Mechanism

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The American Journal of Pathology, Vol. -, No. -, - 2020

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An RGDKGE-Containing Cryptic Collagen Fragment Regulates Phosphorylation of Large Tumor Suppressor Kinase-1 and Controls Ovarian Tumor Growth by a Yes-Associated ProteineDependent Mechanism Xiang H. Han, Jennifer M. Caron, Christine W. Lary, Pradeep Sathyanarayana, Calvin Vary, and Peter C. Brooks From the Maine Medical Center Research Institute, Center for Molecular Medicine, Scarborough, Maine Accepted for publication November 17, 2020. Address correspondence to Peter C. Brooks, Ph.D., Maine Medical Center Research Institute, Center for Molecular Medicine, 81 Research Dr., Scarborough, ME 04074. E-mail: [email protected].

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The growth and spread of malignant tumors, such as ovarian carcinomas, are governed in part by complex interconnected signaling cascades occurring between stromal and tumor cells. These reciprocal cross-talk signaling networks operating within the local tissue microenvironment may enhance malignant tumor progression. Understanding how novel bioactive molecules generated within the tumor microenvironment regulate signaling pathways in distinct cellular compartments is critical for the development of more effective treatment paradigms. Herein, we provide evidence that blocking cellular interactions with an RGDKGE-containing collagen peptide that selectively binds integrin b3 on ovarian tumor cells enhances the phosphorylation of the hippo effector kinase large tumor suppressor kinase-1 and reduces nuclear accumulation of yes-associated protein and its target gene c-Myc. Selectively targeting this RGDKGE-containing collagen fragment inhibited ovarian tumor growth and the development of ascites ﬂuid in vivo. These ﬁndings suggest that this bioactive collagen fragment may represent a previously unknown regulator of the hippo effector kinase large tumor suppressor kinase-1 and regulates ovarian tumor growth by a yes-associated proteinedependent mechanism. Taken together, these data not only provide new mechanistic insight into how a unique collagen fragment may regulate ovarian cancer, but in addition may help provide a potentially useful new alternative strategy to control ovarian tumor progression based on selectively disrupting a previously unappreciated signaling cascade. (Am J Pathol 2020, -: 1e19; https://doi.org/10.1016/j.ajpath.2020.11.009)

It is well established that the stromal microenvironment, which is composed of a variety of diverse cell types, secreted factors, and a complex interconnected network of extracellular matrix (ECM) molecules, can exert profound effects on tumors.1e3 Cellular interactions with speciﬁc ECM components, such as collagen, play central roles in controlling many protumorigenic signaling programs that govern cellular proliferation, survival, migration, and invasion. Dysregulation of these processes underlies many of the altered phenotypic characteristics observed in malignant tumors.4e6 Our laboratory has been focused on developing a more detailed cellular and molecular understanding of how both stromal and tumor cells utilize signaling cascades that are uniquely stimulated by cellular interactions with cryptic or hidden regions within ECM proteins.

Our previous studies have identiﬁed several matriximmobilized cryptic integrin binding sites within ECM molecules that actively regulate angiogenesis, tumor growth, and metastasis.7e9 A variety of studies suggest that, in addition to structurally altered ECM proteins that remain immobilized within the insoluble matrix, bioactive Supported in part by NIH grant CA196739 (P.C.B.) and by the Ofﬁce of Q5 the Assistant Secretary of Defense for Health Affairs, through the Ovarian Cancer Research Program, under award W81XWH-17-1-0099 (P.C.B.). Further support was from the Northern New England Clinical and Translational Research Center grant U54GM115516 (Dr. Clifford Rosen) and the Mesenchymal and Neural Regulation of Metabolic Networks grant P20GM121301 (Dr. Lucy Liaw). Additional support was provided by the Maine Medical Center. Q29 Disclosures: P.C.B. and C.V. hold equity positions in CryptoMedix, Inc.

Copyright ª 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.ajpath.2020.11.009

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fragments of ECM proteins can also be released in a soluble form.10e12 Elevated levels of some of these peptides have been shown to correlate with a more aggressive tumor phenotype in human subjects.12 In fact, new studies have now shown that elevated levels of a deﬁned subset of soluble collagen fragments detected in a cohort of cancer patients correlated with resistance to antieCTLA-4 immune checkpoint therapy.13 These and many other studies are consistent with the possibility that a subset of soluble ECM fragments may not only represent clinically useful biomarkers for studying disease progression and resistance to therapy, but may also play fundamental roles in controlling angiogenesis, inﬂammation, and tumor growth. In this regard, we previously identiﬁed an endogenously expressed soluble collagen fragment generated by a subset of M2-like macrophages.11 This soluble RGDKGE-containing collagen fragment stimulated angiogenesis and inﬂammation in vivo.11 These observations were surprising given that many studies have shown that RGD-containing peptides often exhibit the opposite effect, and inhibit angiogenesis and tumor growth.14,15 This unexpected pro-angiogenic activity appeared to depend in part on the unique ﬂanking sequences surrounding the core RGD motif, as other RGDcontaining collagen peptides lacking this motif failed to signiﬁcantly promote angiogenesis or inﬂammation in this model.11 Although this RGDKGE-containing collagen fragment selectively bound to vascular endothelial cells and stimulated angiogenesis that was dependent in part on the transcriptional coactivator yes-associated protein (YAP), it is not known whether this collagen fragment is generated in malignant tumors or whether it plays a functional role in tumor growth in vivo. Although studies have implicated YAP in promoting the growth of different tumor types, including ovarian carcinomas,16,17 other studies have suggested that YAP signaling might exhibit tumor suppressive activity, depending on the speciﬁc cell type and tissue microenvironment.18,19 In fact, recent studies suggest that YAP activity in peritumoral hepatocytes may suppress the growth of liver tumors.19 Thus, a more indepth molecular understanding of how YAP activity is controlled in context-dependent pathologic processes may allow for the development of innovative new approaches to selectively regulate YAP within distinct tissue microenvironments. A wide array of molecular regulators and mechanisms have been identiﬁed that govern the activation and nuclear accumulation of YAP. Among the mechanisms by which nuclear YAP accumulation is thought to be controlled include the canonical hippo signaling cascade.20,21 Although the ability of the core kinases within the hippo signaling cascade to control YAP activity is well documented, little is known concerning the possibility that soluble collagen-derived fragments may regulate the hippo pathway or whether targeting these extracellular regulators may be exploited to selectively control YAP activity during tumor growth in vivo. Herein, we provide

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evidence that blocking a soluble RGDKGE-containing collagen fragment can regulate the large tumor suppressor kinase-1 (LATS1), a core effector molecule within the hippo pathway. We show that ovarian cancer cell interactions with an RGDKGE collagen peptide activate b3 integrin and reduce the level of inhibitory phosphorylation of YAP and enhance its nuclear accumulation. Moreover, selectively blocking cellular interactions with the RGDKGE collagen peptide enhanced the phosphorylation of LATS1 and YAP, reduced nuclear accumulation of YAP, and reduced the levels of the YAP target gene c-Myc. More important, selectively blocking cellular interactions with the RGDKGE-containing collagen fragment inhibited the development of ascites ﬂuid and ovarian tumors growing in vivo. These ﬁndings indicate that a soluble RGDKGE-containing collagen fragment may represent a previously unknown regulator of the hippo effector kinase LATS1 that plays a role in controlling ovarian tumor growth. These data not only provide new mechanistic insight into how an endogenously generated bioactive collagen fragment may regulate ovarian cancer, but also provide an alternative strategy for the development of new therapeutic approaches to control ovarian tumor progression.

Materials and Methods Cells and Cell Culture Murine ID8evascular endothelial growth factor (VEGF) Q9 ovarian carcinoma cells22 were obtained from Dr. Philip Greenberg (University of Washington School of Medicine, Seattle, WA). ID8-VEGF cells were cultured in Dulbecco’s modiﬁed Eagle’s medium in the presence of 4% fetal bovine serum, 1.0% penicillin-streptomycin, 1.0% sodium pyruvate, and 1% of insulinetransferrinesodium selenite media supplements. SKOV-3 ovarian carcinoma cells were obtained from ATCC (Manassas, VA) and cultured in RPMI 1640 medium in the presence of 5% fetal bovine serum, 1% penicillin-streptomycin, and 1% sodium pyruvate. RAW 264.7 cells were obtained from ATCC and cultured in Dulbecco’s modiﬁed Eagle’s medium in the presence of 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% sodium pyruvate.

Cell Culture in 3D Type I Collagen Gels Brieﬂy, ID8-VEGF cells were mixed with 1 mL of threedimensional (3D) collagen gel-forming solution (Pure Col EZ 3D Type I Collagen Gel Solution; Advanced Biomatrix, San Diego, CA) and incubated for 1 hour at 37 C until gel formation. After adding 1 mL of serum-free Dulbecco’s modiﬁed Eagle’s medium on the top of 3D collagen gel, cells were incubated for 72 hours and the conditioned medium was collected for Western blot analysis.

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Animals

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C57BL/6J female mice (6 to 8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME). NCRNU-F female mice (6 to 8 weeks old) were obtained from Taconic Biosciences (Germantown, NY). Mice were housed in Maine Medical Center Research Institute (Scarborough, ME) pathogen-free air barrier facility, and all animal handling and procedures were approved by the Maine Medical Center Institutional Animal Care and Use Committee.

Reagents, Chemicals, and Antibodies

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Bovine serum albumin, insulinetransferrinesodium selenite media supplements, and crystal violet were obtained from Sigma (St. Louis, MO). Anti-YAP1, antiephosphorylated YAP (Ser127), anti-LATS1, antiephosphorylated LATS (Thr1079), anti-SRC, antiephosphorylated SRC (Y416), antiephosphorylated MST1-Thr-183, antieintegrin b3, and antieTATA-binding protein antibodies were all obtained from Cell Signaling (Danvers, MA). Antiephosphorylated integrin b3 (Ser747) and antieb-actin were obtained from Santa Cruz Biotechnology (Dallas, TX). Anti-MST1 antibody was obtained from Novus Biologicals (Centenial, CO). AntieCD-31 antibody was obtained from Abcam (Cambridge, MA). Monoclonal antibody (Mab) XL313 and nonspeciﬁc control antibody were obtained from Bio X Cell (Lebanon, NH). P2 peptide (CQGPGRDKGEC) and control peptide (CP; CQGPGGAAGGC) were from QED Bioscience (San Diego, CA). Alexa 594elabeled secondary antibody was from Invitrogen (Carlsbad, CA). The SRC inhibitor dasatinib was from Selleckchem (San Diego, CA). Pure Col EZ 3D Type I Collagen Gel Solution was obtained from Advanced Biomatrix. Cell proliferation assay kit was obtained from EMD Millipore (Temecula, CA). Poly-Llysine was obtained from ScienCell Research Laboratories (Carlsbad, CA). Formaldehyde was obtained from Polysciences Inc. (Warrington, PA).

was performed using downloaded ImageJ software version 5.0 (NIH, Bethesda, MD; http:/imagej.nih.gov/ij, last accessed October 6, 2020).9

Nuclear Protein Extraction Subconﬂuent ID8-VEGF cells were washed and serum starved for 1 hour at 37 C, then seeded on 6-well plates coated with CP (100 mg/mL), RGDKGE-containing peptide (P2; 100 mg/mL), or poly-L-lysine (100 mg/mL). In other experiments, subconﬂuent ID8-VEGF cells were stimulated with CP (50 ng/mL) or RGDKGE-containing peptide P2 (50 ng/mL) in suspension for 1 hour at 37 C. In a ﬁnal set of experiments, ID8-VEGF cells were washed and serum starved for 1 hour at 37 C, and cells were then seeded on 6-well plates coated (100 mg/mL) with CP or RGDKGEcontaining peptide (P2). To examine the effects of blocking cellular interactions with the RGDKGE-containing collagen peptide P2 on nuclear accumulation of YAP, cells were seeded on P2 peptide (100 mg/mL)ecoated 6well plates that were preblocked with 100 mg/mL of Mab XL313, antieintegrin b3 antibody, or non-speciﬁc control antibody for 30 minutes. To examine the effects of Src on YAP, ID8-VEGF cells from subconﬂuent culture were washed and serum starved for 30 minutes at 37 C, and cells were pretreated with Src inhibitor dasatinib (1 mmol/L) or dimethyl sulfoxide for 30 minutes. Next, cells were seeded on P2 peptide-coated 6-well plates in presence or absence or Mab XL313 antibody or control antibody. After incubating for 1 hour, adherent and nonadherent cells were harvested. The nuclear and cytoplasmic proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents from Thermo Scientiﬁc (Waltham, MA), according to the manufacturer’s instructions. The nuclear extractions were examined by Western blot analysis with anti-YAP or antieTATA-binding protein antibody, and the cytoplasmic extraction was examined by Western blot analysis with total and phosphorylated YAP (Ser-127), total and phosphorylated LATS1 (Thr-1079), or total and phosphorylated MST1 (Thr-183) antibodies.

Western Blot Analysis RNA Isolation, cDNA Synthesis, and Quantitative PCR

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ID8-VEGF or SKOV-3 cells from subconﬂuent culture were serum starved in suspension for 1 hour at 37 C, and seeded on CP or RGDKGE-containing peptide (P2) coated plates in the presence or absence of Mab XL313 or control antibody (5 mg/mL) for 5 or 15 minutes under the deﬁned experimental conditions and then lysed in radioimmunoprecipitation assay lysis buffer with 1 protease inhibitor cocktail, 2 mmol/L of phenylmethylsulfonyl ﬂuoride, and 1 mmol/L of sodium orthovanadate (Santa Cruz, CA). Equal amounts of cell lysate were separated by SDSPAGE. Membranes were probed with the indicated antibodies. Western blot analyses were performed at least three to four times and visualized by chemiluminescence detection. Quantiﬁcation of the mean relative changes in proteins

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Subconﬂuent ID8-VEGF cells were washed and serum starved for 1 hour at 37 C, and cells were seeded on 6-well plates coated with CP or RGDKGE-containing peptide P2 (100 mg/mL). To examine the effect of blocking cellular interactions with the RGDKGE-containing peptide P2 on c-Myc, cells were washed and serum starved for 30 minutes at 37 C and preblocked with 100 mg/mL of control or Mab XL313 antibodies. Next, cells were seeded on P2 peptide (100 mg/mL)ecoated 6-well plates. After 1-hour incubation, cells were lysed using RNA isolation lysis buffer, and RNAs were isolated by using RNase plus mini kit (Qiagen, Carol Stream, IL) following the manufacturer’s protocol. Total RNA (1.0 mg) was synthesized with iScript cDNA

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synthesis kit from Bio-Rad (Hercules, CA). Equivalent amount of cDNA was used for RT-qPCR, and the murine RT-qPCR primers used for mouse c-Myc were 50 CAGAGGAGGAACGAGCTG-30 (forward) and 50 -TTATG CACCAGAGTTTCGAAGCTGTTCGT-30 (reverse); and the primers used for the housekeeping gene mouse b2m were 50 -CTGACCGGCCTGTATGCTAT-30 (forward) and 50 -CCGTTCTTCCAGCATTTGGAT-30 (reverse). RT-qPC R assays were repeated ﬁve times.

Quantiﬁcation of Tumor Angiogenesis and Levels of Nuclear YAP Frozen sections from individual tumors (N Z 5 per condition) were ﬁxed in 50% methanol and 50% acetone, and blocked with 2.0% bovine serum albumin for 1 hour at room temperature. For quantiﬁcation of angiogenesis, frozen sections of tumors from SKOV-3 or ID8-VEGF ovarian tumors were stained with anti-CD31 (1:300) overnight, followed by incubation with Alexa 594elabeled secondary antibody. Tumor vessel counts were performed from ﬁve to eight 200 microscopic ﬁelds from each of four to ﬁve independent tumors from each condition. For quantiﬁcation of the effect of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc control antibody on nuclear Yap within the ovarian tumors, frozen sections from SKOV-3 or ID8-VEGF ovarian tumors were ﬁxed with 4% formaldehyde at room temperature for 15 minutes. Tumor sections were washed three times in 1 phosphate-buffered saline for 5 minutes each and then blocked with 5% normal goat serum in 3% bovine serum albumin/phosphate-buffered saline for 1 hour. Tissue sections were next stained with anti-YAP (1:100) overnight, followed by incubation with Alexa 594elabeled secondary antibody. Nuclear and cytosolic Yap-positive cell counts were performed from four to nine 400 microscopic ﬁelds from each of four to ﬁve independent tumors from each condition.

Cell-Binding Assays Forty-eightewell nontissue culture plates were coated with 100 mg/mL of CP or RGDKGE-containing peptide (P2). Tumor cells were suspended in binding buffer (RPMI 1640 medium containing 1 mmol/L MgCl2, 0.2 mmol/L MnCl2, and 0.5% bovine serum albumin), and 1 105 cells were added to the wells in the presence or absence of 100 mg/mL of Mab XL313, antieintegrin b3 antibody, and antieintegrin b1 or non-speciﬁc normal mouse IgG control antibody and allowed to bind for 30 to 45 minutes at 37 C. Nonattached cells were removed, and attached cells were stained with crystal violet.7 Cell binding was quantiﬁed by measuring the OD of eluted dye.7 Cell-binding assays were performed at least three times with triplicate wells per condition.

Cell Growth Assays Brieﬂy, ID8-VEGF cells from subconﬂuent culture were serum starved in suspension for 1 hour at 37 C, and seeded 2000 cells per well in 96-well microplate in the presence or absence of Mab XL313 (100 mg/mL) or control antibody (100 mg/mL). After incubating the cells for 72 hours, 10 mL per well WST-1/ECS solution was added to each well and Q15 incubated for 1 hour, and the absorbance was measured by using a microplate reader at 450 nm.

Viral Vectors and Transduction Lentiviral vectors (pLKO.1 based) encoding shRNAs speciﬁc to YAP or a control nontargeting construct developed by the RNAi Consortium were obtained from GE Healthcare Life Science (Lafayette, CO). The effective targeting sequence of YAP gene was 50 -TTCTTTATCTAGCTTGGTGGC-30 . Lentivirus was packaged in the re- Q16 combinant viral vector core facility at Maine Medical Center Research Institute. Cells transduced with the shRNA viruses were selected in growth medium containing 2.5 mg/mL puromycin (Invitrogen) to establish cell pools stably expressing shRNA.

Preparation of Tissue Lysates SKOV-3 and ID8-VEGF ovarian tumors (N Z 4 per tumor type) were harvested from nude mice or C57BL/6 mice at day 14 or day 7, respectively. Individual tumors were snap frozen on dry ice and ground in a cold mortar. Ground up tissues from individual tumors were next mixed with radioimmunoprecipitation assay lysis buffer, and whole tissue lysates were generated. In further experiments, 6- to 8-weekeold C57BL/6J female mice (N Z 4) were scariﬁed for skin and peritoneum tissue harvest. Individual skin or peritoneum tissue was snap frozen on dry ice and ground in a cold mortar with radioimmunoprecipitation assay lysis buffer, and whole tissue lysates were generated.

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Tumor Growth Assays Tumor growth assays were performed as previously described.9 Brieﬂy, mice were injected subcutaneously with either 10 106 ID8-VEGF cells (N Z 8 to 10 C57BL/6 mice per condition) or 3 106 SKOV-3 cells, nontarget SKOV-3 cells, or YAP knockdown SKOV-3 cells (N Z 8 to 10 NCRNU-F mice per condition). Tumors were allowed to form palpable tumors for 3 days, then the mice were injected (intraperitoneally) with Mab XL313 (25 to 250 mg per mouse) or non-speciﬁc control antibody (250 mg per mouse) three times per week for either 42 days for ID8-VEGF tumor model or 35 days for the SKOV-3 tumor model. Tumor size was measured with

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Han et al 621 caliper, and the tumor volume was calculated using the RGDKGE collagen fragment were detected in two of the 622 following formula: Volume Z Length2 Width/2. For four skin samples examined, little if any was detected in any 623 ascites tumor assays, 10 106 ID8-VEGF cells were of the peritoneum tissues (Figure 1B). These data suggest 624 injected (intraperitoneally) in C57BL/6J female mice minimal and differential expression of the 16-Kd RGDKGE 625 (N Z 8). Four weeks later, mice were intraperitoneally collagen fragment within distinct normal tissues. Next, we 626 injected with Mab XL313 or control antibody (100 mg per assessed whether we could detect the 16-Kd RGDKGE 627 mouse) twice a week. At the end of the 13-week treatment collagen fragment in conditioned medium derived from 628 period, ascites ﬂuid was harvested and the volume was ID8-VEGF tumor cells growing in 3D collagen gels. As 629 quantiﬁed. To estimate tumor burden within the diashown in Figure 1C, consistent with our previous ﬁnd630 631 phragm, mouse diaphragms were harvested and wet tissue ings,11 the 16-Kd RGDKGE collagen fragment was readily 632 weights were determined (N Z 5 per group). detected within conditioned medium from M2-like macro633 phages (RAW 264.7), whereas none was detected in cell634 free medium derived from 3D collagen gels under idenStatistical Analysis 635 tical incubation conditions (Figure 1C). Interestingly, the 636 16-Kd RGDKGE collagen fragment was also detected Statistical analysis was performed using the Prism/Graph 637 within conditioned medium derived from ID8-VEGF tumor Q17 Pad statistical program for Macintosh computers. Data were 638 cells grown within 3D collagen gels (Figure 1C). Given analyzed for statistical signiﬁcance using t-test. P < 0.05 639 these ﬁndings and the fact that the 16-Kd RGDKGE 640 was considered signiﬁcant. collagen fragment was detected in solid tumors growing 641 in vivo, we next examined whether ovarian tumor cells 642 Results 643 could directly express the 16-Kd RGDKGE collagen frag644 ment. To this end, whole cell lysates from ovarian tumor Detection of an RGDKGE-Containing Collagen Fragment 645 cell lines were examined. Consistent with our previous in Ovarian Tumors and Interaction with Ovarian 646 studies, the 16-Kd RGDKGE collagen fragment was 647 Carcinoma Cells detected in lysates from RAW 264.7 macrophages 648 (Figure 1D). Interestingly, although low but detectable 649 Our previous studies identiﬁed an endogenously generated levels of the 16-Kd RGDKGE collagen fragment were 650 cryptic collagen fragment containing the amino acid detected in lysates from SKOV-3 tumor cells, relatively 651 sequence RGDKGE that could be detected as an approxihigher levels were detected in ID8-VEGF cell lysates 652 Q18 mately 16-Kd soluble fragment in serum-free conditioned (Figure 1D). Taken together, these data suggest that ovarian 653 11 medium from M2-like macrophages. Given these studies, tumors may represent a biologically relevant tumor type to 654 we sought to determine whether this collagen fragment 655 study the possible impact this collagen fragment might have might be generated during tumor growth. To identify 656 on tumor growth. potentially relevant tumor models with which to study this 657 Although our previous studies indicated that vascular collagen fragment, we examined whole tissue lysates of 658 endothelial cells could directly bind to the RGDKGE SKOV-3 and ID8-VEGF ovarian tumors growing in mice 659 collagen fragment,11 it is not known whether ovarian 660 for the generation of the 16-Kd RGDKGE-containing tumor cells themselves also have this binding ability. 661 ½F1 collagen fragment. As shown in Figure 1A, the 16-Kd Therefore, to further study potential roles of this collagen 662 RGDKGE-containing collagen fragment was detected fragment might have in ovarian cancer, we assessed 663 within whole tumor lysates from both SKOV-3 and ID8whether ovarian carcinoma cells have the capacity to 664 VEGF tumors growing in vivo. To further examine the directly interact with a synthetic RGDKGE-containing 665 expression of this 16-Kd RGDKGE-containing collagen collagen peptide we have called P2.11 As shown in 666 fragment in normal mouse tissues, we examined whole 667 Figure 1, E and F, SKOV-3 and ID8-VEGF tumor cells tissue lysates of skin or peritoneum tissues for individual 668 readily bound to the P2 collagen peptide, while exhibiting mice without tumors. Although variable levels of the 669 670 671 Figure 1 Detection of the RGDKGE-containing collagen fragment in ovarian tumors and its binding to tumor cells. A: Whole tumor lysates were prepared 672 from individual human SKOV-3 (top panels) or murine ID8evascular endothelial growth factor (VEGF; bottom panels) tumors grown in mice. Example of 673 Western blot analysis for the levels of the 16-Kd RGDKGE-containing collagen fragment or loading control actin. B: Whole tissue lysates were prepared from normal skin and peritoneum tissues from individual mice. Western blot analysis for the levels of the 16-Kd RGDKGE-containing collagen fragment or loading 674 control actin. C: Western blot analysis of conditioned medium from RAW 264.7 macrophages, control medium derived from three-dimensional (3D) gel in 675 absence of cells (lane 1), or conditioned medium from ID8-VEGF cells cultured in 3D collagen gels (lanes 2 and 3) for the 16-Kd RGDKGE-containing collagen 676 fragment. D: Western blot analysis of whole cell lysates from RAW 264.7 macrophages or SKOV-3 and ID8-VEGF ovarian carcinoma cells for the 16-Kd RGDKGE677 containing collagen fragment or loading control actin. E: Quantiﬁcation of SKOV-3 cell binding to control peptide (CP) and collagen peptide P2. F: Quan678 tiﬁcation of ID8-VEGF cell binding to CP and collagen peptide P2. G: Quantiﬁcation of SKOV-3 cell binding to collagen peptide P2 in the presence of 679 non-speciﬁc control antibody (Ab Cont) or anti-RGDKGE antibody [Anti-XL313 Monoclonal Antibody (Mab)]. H: Quantiﬁcation of ID8-VEGF cell binding to 680 collagen peptide P2 in the presence of non-speciﬁc Ab Cont or Anti-XL313 Mab. Data represent means SEM (EeH). N Z 4 per group (A and B); N Z at least 681 3 independent experiments (EeH). *P < 0.05. 682

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Han et al 869 minimal if any binding with the non-speciﬁc CP. More Interestingly, our previous studies suggested that the 870 important, Mab XL313, speciﬁcally directed to the RGDKGE collagen peptide could stimulate enhanced nu871 RGDKGE-containing peptide P2, inhibited cellular bindclear accumulation of YAP in endothelial cells.11 Given the 872 ing to P2 compared with a non-speciﬁc control antibody ability of the RGDKGE collagen fragment to bind and 873 (Figure 1, G and H). These data indicate that SKOV-3 and activate b3-integrin in ovarian carcinoma cells, we sought to 874 ID8-VEGF tumor cells can interact with the RGDKGE determine whether b3-integrin receptor may also play a role 875 collagen fragment and may represent useful tumor models in controlling P2 peptide-stimulated nuclear YAP accumu876 with which to study its possible functions. lation in tumor cells. In this regard, we stimulated ID8877 VEGF cells with P2 collagen peptide and examined the 878 879 effects that a function-blocking anti-b3 integrin antibody 880 had on nuclear YAP accumulation. As shown in Figure 2, H The RGDKGE-Containing Collagen Fragment Activates 881 and I, blocking P2 peptide cellular interactions with ID8b3 Integrin and Stimulates Enhanced Nuclear 882 VEGF tumor cells by incubation with anti-b3 integrin Accumulation of YAP 883 antibody signiﬁcantly reduced the relative levels of nuclear 884 YAP. Taken together, these data provide the ﬁrst evidence To determine potential receptors capable of mediating 885 that blocking b3 integrin-mediated interactions with the cellular interactions with the RGDKGE-containing collagen 886 RGDKGE-containing collagen peptide P2 inhibits nuclear peptide P2, cell binding assays were performed in the 887 YAP accumulation in these ovarian carcinoma cells. presence of function-blocking anti-integrin antibodies. As 888 889 ½F2 shown in Figure 2, A and B, function-blocking antibodies 890 directed to b3 integrin signiﬁcantly inhibited binding of The RGDKGE-Containing Collagen Fragment Regulates 891 both SKOV-3 and ID8-VEGF tumor cells to the P2 collagen YAP Phosphorylation in Ovarian Tumor Cells 892 peptide by >80%, whereas a function-blocking antibody 893 directed to b1 integrins had no signiﬁcant inhibitory activity Previous studies have implicated integrin signaling in 894 (Figure 2, A and B). These data indicate that b3 integrin can regulating the activity and nuclear accumulation of YAP in 895 serve as a cell surface receptor for the P2 collagen peptide in different cell types.11,24e26 However, YAP can be 896 these ovarian tumor cells. controlled by diverse mechanisms, and depending on the 897 Next, we examined whether cellular interactions with the particular cell type, may have opposing functions on tumor 898 P2 collagen peptide were sufﬁcient to initiate activation of growth.16,18,19 Therefore, we examined in more detail the 899 b3 integrin, as indicated by phosphorylation of b3 at tyroeffects of stimulation of ovarian tumor cells with the 900 sine 747, which is known to facilitate downstream signaling RGDKGE collagen peptide P2 and its ability to alter nuclear 901 902 events.23 To study this possibility, we focused on accumulation of YAP. As shown in Figure 3A, ID8-VEGF ½F3 903 ID8-VEGF cells, given their relatively high levels of b3 tumor cell binding to P2 resulted in signiﬁcantly enhanced 904 integrin (Figure 2C). To this end, ID8-VEGF cells were levels of nuclear localized YAP. To provide evidence that 905 stimulated by allowing tumor cells to bind to the P2 the enhanced nuclear accumulation of YAP following 906 collagen peptide or a non-speciﬁc CP, and cell lysates were binding to P2 was speciﬁc and not a result of simple me907 prepared. As shown in Figure 2, D and E, tumor cell binding chanical adhesion, YAP nuclear accumulation was 908 to the P2 collagen peptide signiﬁcantly enhanced tyrosine compared between cells binding to P2 or through non909 phosphorylation of b3 integrin by approximately 50%. In integrin interactions with poly-L-lysine. More important, 910 contrast, blocking binding of P2 to the ovarian tumor cells poly-L-lysine supported ID8-VEGF cell binding similarly to 911 with Mab XL313 inhibited b3 integrin phosphorylation P2 (Supplemental Figure S1A). As shown in Figure 3B, 912 (Figure 2, F and G). These data indicate that the P2 collagen tumor cell binding to P2 signiﬁcantly enhanced nuclear 913 914 peptide can directly regulate b3-integrin activation, whereas accumulation of YAP compared with cells binding to poly915 Mab XL313 selectively inhibits this b3-integrin activation. L-lysine. Similarly, stimulation of tumor cells in suspension 916 917 918 Figure 2 The RGDKGE-containing collagen fragment activates b3 integrin and enhances the levels of nuclear yes-associated protein (YAP). A: Quantiﬁ919 cation of SKOV-3 cell binding to collagen peptide P2 in the presence of non-speciﬁc control antibody (NT), function-blocking antieb3-integrin antibody (Anti920 b3), or function-blocking antieb1-integrin antibody (Anti-b1). B: Quantiﬁcation of ID8evascular endothelial growth factor (VEGF) cell binding to collagen 921 peptide P2 in the presence of NT, function-blocking Anti-b3, or function-blocking Anti-b1. C: Western blot analysis of whole cell lysates of ID8-VEGF cells (left panels) and SKOV-3 cells (right panels) for the expression of integrin b3 or actin. D: Whole cell lysates were prepared from ID8-VEGF cells stimulated with 922 control peptide (CP) or collagen peptide P2 for 5 minutes. Example of Western blot analysis for the levels of the phosphorylated b3 integrin (p-b3 Integrin; Tyr 923 747), total b3 integrin (T-b3 Integrin), or loading control actin. E: Quantiﬁcation of the mean fold change in phosphorylated b3 integrin. F: Whole cell lysates 924 were prepared from ID8-VEGF cells stimulated with collagen peptide P2 in presence of non-speciﬁc control antibody (Ab Cont) and anti-RGDKGE collagen 925 fragment antibody [monoclonal antibody (Mab) XL313]. Example of Western blot analysis for the levels of the p-b3 Integrin), T-b3 Integrin, or loading control 926 actin. G: Quantiﬁcation of the mean fold change in phosphorylated b3 integrin. H: Nuclear fractions were prepared from ID8-VEGF cells stimulated with 927 collagen peptide P2 in presence of non-speciﬁc Ab Cont and Anti-b3. Example of Western blot analysis for the levels of nuclear YAP or loading control 928 TATA-binding protein (TBP). I: Quantiﬁcation of the mean fold change in nuclear YAP. Data represent means SEM (A, B, E, G, and I). N Z at least 3 929 independent experiments (E, G, and I). *P < 0.05. 930

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with soluble P2 peptide also signiﬁcantly enhanced accumulation of nuclear localized YAP compared with control (Figure 3C). More important, efﬁcient nuclear and cytoplasmic separation of the samples under the indicated binding conditions was conﬁrmed, as little, if any, b-tubulin was detected in nuclear fractions, but was clearly detected in cytoplasmic fractions, whereas TATA-binding protein was clearly detected in nuclear fractions, whereas little, if any, was observed in cytoplasmic fractions (Figure 3D). Finally, treatment with Mab XL313, but not control antibody, inhibited nuclear YAP levels (Figure 3, E and F). Nuclear localized YAP can interact with transcription factors, such as members of TEAD family, to regulate protumorigenic gene expression programs that contribute to tumor growth and metastasis.27,28 Because YAP regulates the expression of protumorigenic target genes that are thought to play a role in ovarian cancer, such as c-Myc,27e30 we examined whether YAP may play a role in regulating the expression of c-Myc in ID8-VEGF tumor cells. To facilitate these studies, we knocked down YAP in ID8-VEGF cells (Figure 3G) using a YAP-speciﬁc shRNA, and the relative levels of c-Myc mRNA were examined. As shown in Figure 3H, knocking down YAP signiﬁcantly reduced the levels of cMyc compared with control transfected cells, conﬁrming the relevance of YAP in regulating c-Myc in these ovarian tumor cells. Given our ﬁndings that cellular interactions with the RGDKGE-containing collagen peptide P2 regulate YAP, we next examined the effects of P2 stimulation on the relative levels of c-Myc mRNA by RT-qPCR. As expected, tumor cell interaction with the RGDKGE collagen fragment signiﬁcantly enhanced c-Myc (Figure 3I), whereas blocking cellular engagement with the RGDKGE collagen fragment reduced c-Myc mRNA levels (Figure 3J). These data are consistent with the ability of RGDKGE collagen fragment to modulate the expression of a known YAP target gene. The differential localization of YAP between the cytoplasm and the nucleus, where it can function as a transcriptional co-activator regulating gene expression programs, is thought to be controlled by multiple mechanisms, including hippo-dependent and hippo-independent signaling events.16,20,21 To uncover possible mechanisms that might contribute to the ability of this collagen peptide to enhance YAP nuclear localization, we examined YAP phosphorylation, because the phosphorylation of YAP on serine-127 has been shown to play a role in limiting the relative levels of active YAP within the nucleus.20,21 Interestingly, ID8-VEGF cell interactions with P2 collagen peptide signiﬁcantly inhibited the levels of YAP serine 127 phosphorylation (Figure 3, K and L), whereas blocking cellular interactions with P2 enhanced YAP phosphorylation (Figure 3, M and N). Collectively, these ﬁndings suggest that cellular interactions with the RGDKGE collagen fragment can enhance nuclear accumulation of YAP in ovarian carcinoma cells and that this differential accumulation of nuclear YAP was associated with alterations in

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YAP phosphorylation and expression of the YAP target gene c-Myc.

The RGDKGE-Containing Collagen Fragment Regulates the Hippo Pathway Effector Kinase LATS1 by an Src-Dependent Mechanism As mentioned above, the nuclear accumulation of YAP is thought to be governed by multiple mechanisms.16,20,21 LATS1 is a well-established effector molecule of the hippo signaling cascade known to phosphorylate YAP, leading to inactivation and reduced nuclear accumulation.20,21,31,32 In this regard, we examined the levels of phosphorylated LATS1 in ID8-VEGF tumor cells following stimulation with P2 collagen peptide. As shown in Figure 4, A and B, stimulation of tumor cells with P2 collagen peptide signiﬁcantly reduced the relative levels of phosphorylated LATS1, whereas similar experiments performed in the presence of Mab XL313 demonstrated a signiﬁcant enhancement of levels of phosphorylated LATS1 on Thr1079 by over threefold following blocking cellular interactions with P2 collagen peptide (Figure 4, C and D). These data are consistent with the ability of Mab XL313 to inhibit nuclear accumulation of YAP that is associated with the regulation of LATS1. Given that both LATS1 and YAP have been suggested to be controlled in part by Src family kinases,33e35 and that Src is a major downstream effector molecule that can be regulated by integrin signaling, we examined whether Src plays a role in the ability of the P2 collagen fragment to control YAP nuclear accumulation. To this end, we ﬁrst examined the relative levels of P2-stimulated nuclear YAP in tumor cells in which Src was inhibited. Brieﬂy, ID8-VEGF cells were ﬁrst pre-incubated with the Src inhibitor dasatinib to conﬁrm that this Src inhibitor could block Src activity in these ID8-VEGF cells (Figure 4E) and then cells were stimulated with P2 and the relative levels of nuclear YAP were quantiﬁed. As shown in Figure 4, F and G, inhibiting Src reduced the relative levels of P2 peptideestimulated nuclear YAP accumulation compared with control. Moreover, inhibiting Src also prevented the ability of Mab XL313 to alter nuclear accumulation of YAP, as Mab XL313 failed to signiﬁcantly reduce the levels of nuclear YAP in Src-inhibited cells (Figure 4, H and I). Finally, we examined the effects of blocking Src on LATS phosphorylation. Interestingly, although blocking Src in these ID8VEGF cells signiﬁcantly enhanced the relative levels of LATS phosphorylation compared with control (Figure 4, J and K), surprisingly, this treatment also reduced the overall levels of LATS (Figure 4J). Although the mechanism by which Src reduced the overall levels of LATS, and enhanced the levels of the phosphorylated LATS in these ID8-VEGF cells, is currently not known, our data indicate an important role for Src in regulating LATS under these experimental conditions.

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Han et al 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240

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Collagen Fragment Regulates Ovarian Tumors 1241 Given that blocking cellular interactions with the P2 in the chick embryo model,11 we examined SKOV-3 tumors 1242 collagen peptide signiﬁcantly enhanced phosphorylated for the relative levels of angiogenesis by quantifying the 1243 LATS1, we next examined the effects that Src inhibition has number of CD31-expressing tumor vessels. Tumors from 1244 on the ability of Mab XL313 to control LATS1 phosphorymice treated with Mab XL313 resulted in signiﬁcantly 1245 lation. As expected, blocking Src inhibited the ability of Mab reduced levels of tumor vessels by approximately 35% 1246 XL313 to regulate LATS1 phosphorylation (Figure 4, L and (Figure 5B). To conﬁrm the antitumor activity of Mab 1247 M). Interestingly, blocking cellular interactions with P2 had XL313 in a second ovarian tumor model, we examined the 1248 no detectable effect on the relative levels of activated hippo effects of Mab XL313 on ID8-VEGF tumor growth. Murine 1249 kinase MST1 (Figure 4N). Taken together, these ﬁndings ID8-VEGF tumor cells were injected subcutaneously, and 1250 1251 suggest that the ability of the RGDKGE collagen fragment to beginning on day 3, mice were treated as before. Although 1252 regulate the hippo effector kinase LATS1 and its downstream these s.c. ID8-VEGF tumors grew slowly and showed evi1253 target YAP in these tumor cells depends on Src. dence of regression irrespective of the treatment, the size of 1254 ID8-VEGF tumors from mice treated with Mab XL313 was 1255 signiﬁcantly reduced by approximately 50% compared with Selective Targeting of the RGDKGE-Containing Collagen 1256 control (Figure 5C). Interestingly, incubation of ID8-VEGF Fragment Inhibits Ovarian Tumor Growth 1257 cells with Mab XL313 had no signiﬁcant inhibitory activity 1258 on cell growth in vitro compared with control antibody The RGDKGE collagen fragment was previously shown to 1259 (Supplemental Figure S1B). However, similar to that play a role in angiogenesis and inﬂammation.11 However, it 1260 observed in SKOV-3 tumors, Mab XL313 signiﬁcantly is not known whether this collagen fragment may impact 1261 inhibited tumor angiogenesis in ID8-VEGF tumors tumor growth in vivo. Given our current observations, we 1262 1263 (Figure 5D). Given our studies indicating that Mab XL313 sought to examine whether selective targeting of this 1264 could reduce the levels of nuclear accumulation of YAP in collagen fragment might regulate ovarian tumor growth. To 1265 ovarian tumor cells in vitro, we examined ovarian tumors examine this possibility, we ﬁrst assessed the dose1266 from mice treated with Mab XL313 for the relative levels of dependent effects of Mab XL313 on ovarian tumors 1267 nuclear localized YAP. As shown in Figure 5, E and F, growing in mice. Human SKOV-3 ovarian tumor cells were 1268 although clear variation in the overall levels of YAP was injected subcutaneously. Once tumors reached a size of 1269 3 observed between SKOV-3 and ID8-VEGF tumors approximately 100 mm , mice were treated with Mab 1270 (Figure 5, E and F), the relative levels of nuclear localized XL313 or non-speciﬁc control antibody three times per 1271 YAP were consistently reduced within tumors from mice 1272 ½F5 week at the doses indicated. As shown in Figure 5A, treated with Mab XL313 compared with control (Figure 5, E treatment with Mab XL313 exhibited dose-dependent 1273 and F). inhibitory activity, and showed a signiﬁcant inhibition of 1274 1275 Ovarian tumors growing within the s.c. space may not approximately 50% on day 35. Given our previous studies 1276 represent an optimal model to study ovarian tumor growth indicating that selectively targeting the RGDKGE collagen 1277 due in part to the lack of a mesothelial cell layer covering fragment could inhibit growth factoreinduced angiogenesis 1278 1279 1280 Figure 3 Regulation of yes-associated protein (YAP) phosphorylation and target gene c-Myc by the RGDKGE collagen fragment. A: Nuclear fractions were 1281 prepared from ID8evascular endothelial growth factor (VEGF) cells stimulated with collagen peptide P2 or control peptide (CP). Top panels: Examples of 1282 Western blot analyses for the levels of nuclear YAP or loading control TATA-binding protein (TBP). Bottom panel: Quantiﬁcation of the mean fold change in 1283 nuclear YAP. B: Nuclear fractions were prepared from ID8-VEGF cells binding to poly-L-lysine (Pol-L) or collagen peptide P2. Top panels: Examples of Western blot analyses for the levels of nuclear YAP or loading control TBP. Bottom panel: Quantiﬁcation of the mean fold change in nuclear YAP. C: Nuclear fractions 1284 were prepared from ID8-VEGF cells binding to soluble collagen peptide P2 or CP in suspension. Top panels: Example of Western blot analyses for the levels of 1285 nuclear YAP or loading control TBP. Bottom panel: Quantiﬁcation of the mean fold change in nuclear YAP. D: Western blot analysis of nuclear and cytoplasmic 1286 fractions prepared from ID8-VEGF cells stimulated with CP, collagen peptide P2, or poly-L-lysine for cytoplasmic marker b-tubulin or the nuclear marker TBP. E: 1287 Nuclear fractions were prepared from ID8-VEGF cells stimulated with collagen peptide P2 in presence of non-speciﬁc control antibody (Ab Cont) or anti-RGDKGE 1288 collagen fragment antibody [monoclonal antibody (Mab) XL313]. Example of Western blot analysis for the levels of nuclear YAP or loading control TBP. F: 1289 Quantiﬁcation of the mean fold change in nuclear YAP from ID8-VEGF cells stimulated with collagen peptide P2 in presence of non-speciﬁc Ab Cont or anti1290 RGDKGE collagen fragment antibody (Mab XL313). G: Western blot analysis of whole cell lysates prepared from control shRNA transfected (NT-shRNA) and Yap1291 shRNA transfected ID8-VEGF ovarian tumor cells for YAP expression or loading control actin. Relative fold change in levels of protein expression was 1292 determined by ImageJ software. H: ID8-VEGF cells were transfected with either a nontargeting control shRNA (Cont-NT) or YAP-speciﬁc shRNA (YAP/KD). 1293 Quantiﬁcation of the mean relative fold change in c-Myc mRNA. I: Quantiﬁcation of the mean fold change in c-Myc mRNA levels in ID8-VEGF cells stimulated with CP or collagen peptide P2. J: Quantiﬁcation of the mean fold change in c-Myc mRNA levels in ID8-VEGF cells stimulated with collagen peptide P2 in the 1294 presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313). K: Western blot analysis of cytosolic fractions prepared from ID81295 VEGF cells stimulated with CP or collagen peptide P2 for the levels of phosphorylated YAP (p-YAP; Ser-127), total YAP (T-YAP), or loading control actin. L: 1296 Quantiﬁcation of the mean fold change in phosphorylated YAP from ID8-VEGF cells stimulated with CP or collagen peptide P2. M: Western blot analysis of 1297 cytosolic fractions prepared from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment 1298 antibody (Mab XL313) for p-YAP, T-YAP, or loading control actin. N: Quantiﬁcation of the mean fold change in p-YAP from ID8-VEGF cells stimulated with 1299 collagen peptide P2 in the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313). Data represent means SEM (AeC, F, 1300 HeJ, L, and N). N Z at least 3 independent experiments (A, bottom panel, F, I, J, L, and N); N Z 3 independent experiments (B and C, bottom panels); 1301 N Z 4 independent experiments (H). *P < 0.05. 1302

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Collagen Fragment Regulates Ovarian Tumors tissues that are typically colonized within the peritoneal cavity, where ovarian tumors typically grow.36,37 Therefore, we next examined the effects of targeting the RGDKGE collagen fragment might have on ID8-VEGF ascites ﬂuid formation and tumor colonization of the diaphragm, which represent a common tissue location of ovarian tumors in mice and humans.36,37 To this end, mice were injected intraperitoneally with ID8-VEGF cells, which were allowed to grow for 4 weeks before initiating treatment. Beginning at 4 weeks, mice were treated with either non-speciﬁc control antibody or Mab XL313 two times per week. At the end of 13 weeks, mice were sacriﬁced and ascites ﬂuid and diaphragms were examined. As shown in Figure 5G, mice treated with control antibody formed ascites, as indicated by extensive distention of the abdominal cavities, whereas mice treated with Mab XL313 exhibited less abdominal distension. Quantiﬁcation of the relative volume of tumor ascites ﬂuid indicated that mice treated with Mab XL313 had signiﬁcantly reduced levels by >60% (Figure 5G). Moreover, quantiﬁcation of tumor burden, as indicated by wet tissue weights of the diaphragms, indicated that mice treated with Mab XL313 exhibited an approximately 30% reduction in wet weight compared with controls (Figure 5H). These novel ﬁndings suggest that the endogenously generated RGDKGE collagen fragment plays a role in ovarian tumor growth in vivo.

Mab XL313eMediated Inhibition of Ovarian Tumor Growth Depends on Altering YAP The transcriptional co-activator YAP plays roles in angiogenesis, tumor growth, and metastasis in multiple tumor models.16,24e28 However, other studies have suggested that YAP may have tumor suppressive functions, depending on the tissue location and cell type.18,19 Our

1551 current studies indicate that the RGDKGE-containing 1552 collagen fragment can bind to integrin b3 and stimulate 1553 a signaling cascade, leading to enhanced nuclear accu1554 mulation of YAP. Given these observations, we sought to 1555 determine whether the antitumor activity of Mab XL313 1556 depends on altering YAP. To facilitate these studies, we 1557 knocked down expression of YAP in SKOV-3 cells using 1558 a YAP-speciﬁc shRNA (Figure 6A), and examined the ½F6 1559 antitumor activity of Mab XL313 on the growth of either 1560 1561 control transfected SKOV-3 cells (NT-SKOV-3) or 1562 SKOV-3 cells in which YAP was knocked down (YAP1563 KD-SKOV-3). Tumor cell variants were injected in mice, 1564 and beginning on day 3, mice were treated three times per 1565 week with 100 mg of Mab XL313 or control antibody. As 1566 shown in Figure 6B, Mab XL313 signiﬁcantly inhibited 1567 the growth of control transfected (NT-SKOV-3) tumors by 1568 approximately 50% on day 35. In contrast, Mab XL313 1569 failed to signiﬁcantly inhibit the growth of SKOV-3 tu1570 mors (YAP-KD-SKOV-3) in which the levels of YAP had 1571 been reduced (Figure 6C). These data are consistent with 1572 1573 notion that the antitumorigenic activity of Mab XL313 1574 depends on inhibiting YAP. Collectively, our data are 1575 consistent with a working model in which ovarian tumor 1576 cell interactions with an endogenously generated 1577 RGDKGE-containing collagen fragment stimulate a b31578 integrin signaling cascade that results in suppressing 1579 phosphorylation of the effecter kinase LATS1, ultimately 1580 leading to enhanced nuclear accumulation of YAP 1581 (Figure 6D). 1582

Discussion Although the ability of the local stromal microenvironment to control tumor growth has been appreciated for years,

Figure 4

Regulation of large tumor suppressor kinase-1 (LATS1) phosphorylation by the RGDKGE collagen fragment. A: Cytosolic fractions were prepared from ID8evascular endothelial growth factor (VEGF) cells stimulated with collagen peptide P2 or control peptide (CP). Example of Western blot analysis for the levels of phosphorylated LATS1 (p-LATS1; Thr 1079) or loading control actin. B: Quantiﬁcation of the mean fold change in p-LATS1 from ID8-VEGF cells stimulated with collagen peptide P2 or CP. C: Cytosolic fractions were prepared from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of nonspeciﬁc control antibody (Ab Cont) or anti-RGDKGE collagen fragment antibody [monoclonal antibody (Mab) XL313]. Example of Western blot analysis for the level of p-LATS1 or loading control actin. D: Quantiﬁcation of the mean fold change in p-LATS1 from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313). E: ID8-VEGF cells were treated (1 mmol/L) with the Src inhibitor dasatinib (Src In) or control [dimethyl sulfoxide (DMSO)]. Representative example of Western blot analysis for phosphorylated Src (p-Src; Tyr 416), total Src (T-Src), or loading control actin. Relative fold change in levels of protein expression was determined by ImageJ software. F: Nuclear fractions were prepared from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of control (DMSO) or Src In. Example of Western blot analysis for the levels of nuclear yes-associated protein (YAP) or loading control TATA-binding protein (TBP). G: Quantiﬁcation of the mean fold change in nuclear YAP from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of control (DMSO) or Src In. H: Western blot analysis of nuclear fractions from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of the Src In and in the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313) for the levels of nuclear YAP or loading control TBP. I: Quantiﬁcation of the mean fold change in nuclear YAP from nuclear fractions from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of the Src In and in the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313). J: Western blot analysis of ID8-VEGF cells treated with DMSO or Src In for p-LATS1 or loading control actin. K: Quantiﬁcation of the mean fold change in p-LATS1 from ID8-VEGF cells treated with DMSO or Src In. L: Western blot analysis of cytosolic fractions from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of the Src In and the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313) for p-LATS1 or loading control actin. M: Quantiﬁcation of the mean fold change in p-LATS1 from ID8-VEGF cells stimulated with collagen peptide P2 in the presence of the Src In and the presence of non-speciﬁc Ab Cont or anti-RGDKGE collagen fragment antibody (Mab XL313). N: Western blot analysis of ID8-VEGF cells incubated in the presence Mab XL313 or non-speciﬁc control antibody seeded on RGDKGE-containing collagen peptide P2 for phosphorylated MST1 (p-MST1; Thr 183), total MST1 (T-MST1), or loading control actin. Relative fold change in levels of protein expression was determined by ImageJ software. Data represent means SEM (B, D, G, I, K, and M). N Z at least 3 independent experiments (B, D, G, I, K, and M). *P < 0.05. T-LATS1, total LATS1.

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Collagen Fragment Regulates Ovarian Tumors effectively translating this cellular and molecular insight into new therapeutic strategies has been slow to emerge, due in part to the complexity of the various functional elements that constitute the tumor microenvironment. Stromal cells known to inﬂuence tumor growth include endothelial cells and a variety of differentially polarized ﬁbroblasts and immune cells.1e3 These diverse groups of cells not only modulate the behavior of solid tumors by secreting bioactive molecules that can alter the structural integrity of the ECM, but they also secrete many soluble factors that directly bind to nearby tumor cells and modulate diverse signaling pathways.1e6,37,38 In a reciprocal manner, stromal cells can also respond to soluble factors released from tumor cells, and these soluble cell-cell communication pathways help support tumor progression.1e6,37,38 This complex cross talk occurring between different cell types and between cells and a variety of ECM components plays critical roles in regulating tumor growth and metastasis, as well as inﬂuencing how tumors respond to different cancer therapies.1e6,37,38 Identifying soluble bioactive molecules that have the capacity to alter the behavior of both stromal and tumor cells will likely provide important new insight into how crosstalk signaling mechanisms are coordinated, and may help with the design of more effective therapeutic strategies to manage malignant tumor progression. We previously identiﬁed a 16-Kd RGDKGE-containing collagen fragment that was actively secreted by M2-like macrophages and stimulated nuclear accumulation of YAP in endothelial cells.11 Although it is well documented that YAP can differentially regulate several distinct gene expression programs in different cell types, the role of YAP in controlling biological processes, such as organ size, stem cell functions, differentiation, and tumor growth, likely depends on how YAP activity is controlled in distinct cell populations within a given tissue microenvironment.39e41 In fact, the precise biological impact of YAP, as it relates to both normal physiological processes as well as diseases,

such as cancer, has been shown to be context dependent.39e41 For example, YAP has been shown to control the size of organs, such as the heart and liver,42e44 whereas loss of YAP had minimal effects or reduced the size of other histologically distinct organs, such as the intestine and pancreas.45e47 Moreover, although reducing YAP activity inhibits the growth of tumors, such as melanoma and lung carcinomas,48,49 other studies suggested that YAP under speciﬁc circumstances may have tumor suppressive functions in breast and liver carcinomas.18,19 These and other studies indicate the need for a more in-depth understanding of the different mechanisms by which YAP is regulated within distinct cell types. Uncovering novel mechanisms by which YAP may be regulated under pathologic conditions may allow for more optimized strategies to control YAP activity in the context of tumor growth. YAP activity can be regulated by diverse mechanisms, including cell adhesion, mechanical forces, stress signaling, and soluble factors,16,20,21 some of which can be altered by the canonical hippo signaling cascade.16,20,21 In addition, YAP can also be regulated by hippo pathwayeindependent mechanisms, such as those involved in altered actin polymerization.16,20,21 Given these facts, coupled with the known functions of YAP in controlling gene expression programs,27,28 deﬁning strategies to selectively control YAP activity under pathologic conditions will likely contribute to optimizing the therapeutic efﬁcacy of targeting YAP. Although intact collagen within the insoluble ECM is thought to regulate ovarian carcinoma progression,36,50 little is known as to whether soluble bioactive collagen fragments regulate ovarian tumor growth in vivo. Herein, we provide evidence, for the ﬁrst time, that a unique RGDKGEcontaining collagen fragment can be detected in ovarian tumors growing in mice and can also be generated by some ovarian tumor cell lines. Interestingly, although variable levels of the RGDKGE-containing collagen fragment were

Figure 5

Inhibition of the RGDKGE collagen fragment inhibits ovarian tumor growth. A: Quantiﬁcation of the dose-dependent effects of anti-RGDKGE collagen fragment antibody [monoclonal antibody (Mab) XL313] or non-speciﬁc control antibody (Ab Cont) on the growth of SKOV-3 tumors in vivo. Arrow indicates start of treatment with antibodies. Data points indicate tumor volumes. B: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont on SKOV-3 tumor angiogenesis. Data bars indicate number of CD31-positive vessels per 200 ﬁeld within ﬁve independent ﬁelds from ﬁve different tumors per condition. C: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or nonspeciﬁc Ab Cont on the s.c. growth of ID8evascular endothelial growth factor (VEGF) tumors in vivo. Data bars indicate tumor volume measured on day 42. D: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont on ID8-VEGF tumor angiogenesis. Data bars indicate number of CD31-positive vessels per 200 ﬁeld within eight independent ﬁelds from ﬁve to six different tumors per condition. E: Tissue sections from SKOV-3 tumors from mice treated with control antibody or Mab XL313 were analyzed for the relative levels of nuclear localized yes-associated protein (YAP). Top panels: Examples of nuclear localized YAP; arrows indicate examples of nuclear accumulation of YAP (pink indicates overlap of blue DAPI and red YAP staining). Bottom panel: Quantiﬁcation of mean percentage nuclear-positive cells per 400 ﬁeld from seven to nine ﬁelds from four independent tumors per condition. F: Tissue sections from ID8-VEGF tumors from mice treated with control antibody or Mab XL313 were analyzed for the relative levels of nuclear localized YAP. Top panels: Examples of nuclear localized YAP; arrows indicate examples of nuclear accumulation of YAP (pink indicates overlap of blue DAPI and red YAP staining). Bottom panel: Quantiﬁcation of mean percentage nuclear-positive cells per 400 ﬁeld from four to seven ﬁelds from ﬁve independent tumors per condition. G: Left panel: Representative example of the differential abdominal distension of ID8-VEGF cell-injected mice treated for 13 weeks with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont. Right panel: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont on the relative volume of ascites. Data bars indicate ascites ﬂuid volume measured at 13 weeks. H: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont on the wet weights of mouse diaphragms. Data bars indicate wet weight measured at 13 weeks. Data indicate means SEM (AeH). N Z 8 per group (A and C); N Z 5 per group (G and H). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar [ 20 mm (E and F).

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Figure 6 Monoclonal antibody (Mab) XL313emediated inhibition of ovarian tumor growth depends on yes-associated protein (YAP). A: Western blot analysis of whole cell lysates prepared from control shRNAetransfected (NT-shRNA) and YAP-shRNAetransfected SKOV-3 ovarian tumor cells for YAP or loading control actin. Relative fold change in levels of protein expression was determined by ImageJ software. B: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc control antibody (Ab Cont) on the growth of control-transfected SKOV-3 tumor cells (nontargeting SKOV-3) in vivo. Arrow indicates start of treatment with antibodies. Data points indicate tumor volumes. C: Quantiﬁcation of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-speciﬁc Ab Cont on the growth of SKOV-3 tumor cells in which YAP levels have been knocked down (YAP/KD-SKOV-3). Arrow indicates start of treatment with antibodies. Data points indicate tumor volumes. D: Working model of a potential mechanism by which the endogenously generated RGDKGE collagen fragment may regulate the nuclear accumulation of YAP. Data indicate means SEM (B and C). N Z 8 per group (B and C). *P < 0.05. LATS1, large tumor suppressor kinase-1.

detected in some normal skin samples from mice, little, if any, of this collagen fragment was detected in normal peritoneal tissues, where ovarian tumors often grow. Given our previous studies and our current data, we sought to determine whether the RGDKGE collagen fragment might also directly bind to ovarian carcinoma cells and regulate

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tumor growth. Our data indicate that SKOV-3 and ID8VEGF ovarian tumor cells can bind to the soluble RGDKGE collagen fragment using b3 integrin as a function-blocking antibody directed to this integrininhibited binding, whereas a function-blocking antibody directed to b1-containing integrins failed to demonstrate any

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Collagen Fragment Regulates Ovarian Tumors effect. Ovarian tumor cell binding to the RGDKGE collagen peptide led to enhanced phosphorylation and activation of b3 integrin, and enhanced nuclear accumulation of YAP, which could be speciﬁcally inhibited by directly targeting the RGDKGE collagen peptide with Mab XL313. More important, the exact molecular mechanism by which Src controls the ability of the RGDKGE collagen fragment to regulate LATS1 and YAP is not completely understood, and work is currently underway to deﬁne this signaling pathway. The precise role of b3 integrin in controlling tumor growth is complex, as studies have provided evidence for both protumorigenic as well as tumor suppressive functions. For example, studies have shown correlations between high expression of b3 integrin and prognosis in patients with melanoma and breast carcinoma.51,52 Other studies using animal models have shown that directly blocking b3 integrin inhibits angiogenesis and tumor growth.14,15,53,54 In contrast, other work examining ovarian carcinomas has shown that high levels of b3 integrin correlated with improved clinical outcome and that reducing b3 integrin expression enhanced ovarian tumor growth.55 These seemingly opposing functions for b3 integrin might be explained, at least in part, by the fact that the ﬁnal outcome of b3 integrin signaling within a particular cell or tumor type depends in part on the nature of the different ligands binding to this integrin, and the types of adaptor molecules that are assembled into signaling complexes that facilitate transmission of extracellular cues. For example, it is well established that certain b3 integrin binding ligands, such as vitronectin, osteopontin, and periostin, can induce protumorigenic signaling events.56e58 In contrast, other bioactive factors that bind to b3 integrin, such as tumstatin, canstatin, and endostatin, have been shown to inhibit tumor growth.59e61 Interestingly, overexpression of b3 integrin in some ovarian tumor cell lines resulted in reduced proliferation that was associated with a G2/M block in the cell cycle and altered phosphorylation of CD2.55 In other studies, evidence suggests that unoccupied integrin avb3 may result in signaling, leading to a process termed integrin-mediated cell death.62 The ability of avb3 to control distinct functions in different cell types, such as immune cells, may also impact the overall contribution of avb3 to tumor growth. For example, avb3 expressed in macrophages and dendritic cells has been shown to play roles in phagocytosis of tumor cells as well as promoting antigen presentation.63,64 In addition, avb3 has also been shown to play a role in regulating T-cell receptor signaling and motility,65,66 all of which may depend on the molecular features of the different ligands that bind to this integrin. In fact, studies have shown that direct targeting of b3 or reducing b3 integrin in a subpopulation of immune cells results in the induction of immune suppression,63,64 thereby compromising the efﬁcacy of this therapeutic strategy as it relates to tumor growth. Therefore, designing and evaluating alternative strategies to disrupt avb3-dependent signaling without

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directly targeting avb3 itself may help in the development of more effective treatments. Our ﬁndings and many other studies are consistent with the notion that the overall contribution of avb3 to tumor growth depends on a complex and interconnected network of parameters that include the cell type that the integrin is functioning in, the molecular characteristics of the local avb3 binding ligands, and the bioavailability and composition of the downstream signaling molecules required for the formation of intracellular signaling complexes that facilitate activation of distinct signaling cascades. Given this complexity, a therapeutic strategy of directly targeting all avb3 in different cell types may not represent an optimal approach. As an alternatively strategy, we have been studying whether selective targeting of discrete protumorigenic ligands of integrin avb3, instead of directly targeting the integrin itself, may provide a clinically effective new approach to control protumorigenic signaling events. This alternative strategy might allow for selective inhibition of protumorigenic stimuli mediated through integrin avb3 and, at the same time, allow for the engagement of locally available tumor suppressing ligands to simulate inhibitory signals through avb3 within distinct stromal and tumor cell populations, ultimately promoting a more robust therapeutic outcome when used either alone or in combination with other cancer treatments. Taken together, a therapeutic strategy that focuses on selectively targeting a novel protumorigenic bioactive ligand of avb3 integrin, rather than directly targeting the avb3 receptor itself, may help selectively disrupt protumorigenic signaling pathways operating within both tumor and stroma cells to allow for an effective treatment paradigm for managing ovarian cancer.

Supplemental Data Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2020.11.009.

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23. Blystone SD: Kinetic regulation of beta 3 integrin tyrosine phosphorylation. J Biol Chem 2002, 277:46886e46890 24. Caron JM, Han X, Contois L, Vary CPH, Brooks PC: The HU177 collagen epitope controls melanoma cell migration and experimental metastasis by a CDK5/YAP-dependent mechanism. Am J Pathol 2018, 188:2356e2368 25. Cosset E, Ilmjarv S, Dutoit V, Elliott K, von Schalscha T, Camargo MF, Reiss A, Moroishi T, Seguin L, Gomez G, Moo JS, Preynat-Seauve O, Krause KH, Chneiweiss H, Sarkaria JN, Guan KL, Dietrich PY, Weis SM, Mischel PS, Cheresh DA: Glut3 addiction is a druggable vulnerability for a molecularly deﬁned subpopulation of glioblastoma. Cancer Cell 2017, 32:856e868 26. Elbediwy A, Vincent-Mistiaen ZI, Spencer-Dene B, Stone RK, Boeing S, Wculek SK, Cordero J, Tan EH, Ridgway R, Brunton VG, Sahai E, Gerhardt H, Behrens A, Malanchi I, Sansom OJ, Thompson BJ: Integrin signaling regulates YAP and TAZ to control skin homeostasis. Development 2016, 143:1674e1687 27. Zhou Y, Huang T, Cheng AS, Yu J, Kang W, To KF: The TEAD family and its oncogenic role in promoting tumorigenesis. Int J Mol Sci 2016, 17:138 28. Zanconato F, Forcato M, Battilana G, Azzolin L, Quaranta E, Bodega B, Rosato A, Bicciato S, Cordenonsi M, Piccolo S: Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat Cell Biol 2015, 17: 1218e1227 29. Tashiro H, Miyazaki K, Okamura H, Iwai A, Fukumoto M: C-MYC over-expression in human primary ovarian tumours: its relevance to tumour progression. Int J Cancer 1992, 50:828e833 30. Prathapam T, Aleshin A, Guan Y, Gray JW, Martin GS: p27Kip1 mediates addiction of ovarian cancer cells to MYCC (c-MYC) and their dependence on MYC paralogs. J Biol Chem 2010, 285: 32529e32538 31. Hao Y, Chun A, Cheung K, Rashidi B, Yang X: Tumor suppressor LATS1 is a negative regulator of oncogene YAP. J Biol Chem 2008, 283:5496e5509 32. St John MA, Tao W, Fei X, Fukumoto R, Carcangiu ML, Brownstein DG, Parlow AF, McGrath J, Xu T: Mice deﬁcient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction. Nat Genet 1999, 21:182e186 33. Si Y, Ji X, Cao X, Dai X, Xu L, Zhao H, Guo X, Yan H, Zhang H, Zhu C, Zhou Q, Tang M, Xia Z, Li L, Cong YS, Ye S, Liang T, Feng XH, Zhao B: Src inhibits the hippo tumor suppressor pathway through tyrosine phosphorylation of Lats1. Cancer Res 2017, 77: 4868e4880 34. Lamar JM, Xiao Y, Norton E, Jiang ZG, Gerhard GM, Kooner S, Warren JSA, Hynes RO: SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis. J Biol Chem 2019, 294:2302e2317 35. Ege N, Dowbaj AM, Jiang M, Howell M, Hooper S, Foster C, Jenkins RP, Sahai E: Quantitative analysis reveals that actin and Src-family kinases regulate nuclear YAP1 and its export. Cell Syst 2018, 6:692e708 36. Natarajan S, Foreman KM, Soriano MI, Rossen NS, Shehade H, Fregoso DR, Eggold JT, Krishnan V, Dorigo O, Krieg AJ, Heilshorn SC, Sinha S, Fuh KC, Rankin EB: Collagen remodeling in the hypoxic tumor-mesothelial niche promotes ovarian cancer metastasis. Cancer Res 2019, 79:2271e2284 37. Lengyel E: Ovarian cancer development and metastasis. Am J Pathol 2010, 177:1053e1064 38. De Nola R, Menga A, Castegna A, Loizzi V, Ranieri G, Cicinelli E, Cormio G: The crowded crosstalk between cancer cells and stromal microenvironment in gynecological malignancies: biological pathways and therapeutic implication. Int J Mol Sci 2019, 20:2401 39. Reggiani F, Gobbi G, Ciarrocchi A, Ambrosetti DC, Sancisi V: Multiple roles and context-speciﬁc mechanisms underlying YAP and TAZ-mediated resistance to anti-cancer therapy. Biochim Biophys Acta Rev Cancer 2020, 1873:188341

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