Supplementary Figure 4. α3 integrin recycled to lamellipodia. Surface α3 integrin was labelled with P1B5 rabbit antibody in adherent DU145 cell monolayer and allowed to internalize for 40 minutes at 37{degree sign}C. Uninternalized label remaining at the cell membrane is detected and blocked from internalization using Alexa 568 conjugated anti-rabbit secondary antibody. P1B5 antibody bound integrin protected inside the cells was allowed to recycle back and subsequently reacted with Alexa 488 conjugated anti-rabbit secondary antibody. (A) Total membrane and internalized intracellular α3 integrin in permeabilized cells shown as control (red). (B) Uninternalized α3 integrin (red) and recycled α3 integrin (green) at 0min, 10min and 40 minutes of recycling and 40min recycling with primaquine (PQ, recycling inhibitor) are shown in merged images with DAPI (blue) stained nucleus (left panel). Middle panel shows only the recycled α3 integrin label in grey. Right panel is magnified images of boxed sections marked for recycled integrin localized at lamellipodia at cell front (white arrows) or at cell- cell locations (closed white triangles) and uninternalized integrin at cell front (open white triangles). Images acquired by deconvolution microscopy and single Z-plane is shown. Bars, 20µm. Images are representative fields of view obtained in 3 independent experiments.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

Supplementary Figure 2. Distribution of α6 integrin in EEA1 vesicles. Surface integrin α6 was labelled with J1B5 in DU145 cells and allowed to internalize for 40 minutes. Cells were fixed, permeabilized, and immunostained for the early endosome marker EEA1. (A) Untreated DU145 cells and (B) siFIP5 treated cells stained for α6 integrin (red), EEA1 (green) and DAPI (blue) in merged images. Images acquired by deconvolution microscopy and single Z-plane is shown. (C) Magnified images of boxed sections for untreated and siFIP5 DU145 cells. White arrows indicate colocalization of α6 integrin in EEA1 vesicles. (D) Percent α6 integrin in EEA1 vesicles and mean Pearson coefficients of correlation of α6 integrin with EEA1 (Pr(α6/EEA1)) for untreated or siFIP5 cells are shown based on 10 different fields of view in 4 independent experiments (*p<0.05).

Supplementary Figure 1. PC3N cells are negative for β4 integrin expression. DU145 and PC3N cells were labelled with an antibody against β4 integrin and cell surface levels of β4 integrin were measured using flow cytometry. Flow histograms of β4 integrin surface levels (top panel) in unlabeled PC3N cells (solid line), PC3N cells labelled with a non-specific fluorophore conjugated secondary antibody (2{degree sign} Ab only, dotted line) and DU145 (shaded solid line) and PC3N (dashed line) cells labeled with antibody against β4 integrin. The mean peak fluorescence values are reported for each condition (bottom panel).

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Enzymatically inactive tissue-type plasminogen activator (EI-tPA) does not activate fibrinolysis, but interacts with the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) in macrophages to block innate immune system responses mediated by toll-like receptors. Herein, we examined the ability of EI-tPA to treat colitis in mice, induced by dextran sulfate sodium. In two separate studies, designed to generate colitis of differing severity, a single dose of EI-tPA administered after inflammation established significantly improved disease parameters. EI-tPA-treated mice demonstrated improved weight gain. Stools improved in character and became hemoccult negative. Abdominal tenderness decreased. Colon shortening significantly decreased in EI-tPA-treated mice, suggesting attenuation of irreversible tissue damage and remodeling. Furthermore, histopathologic evidence of disease decreased in the distal 25% of the colon in EI-tPA-treated mice. EI-tPA did not decrease the number of CD45-positive leukocytes or F4/80-positive macrophage-like cells detected in extracts of colons from dextran sulfate sodium-treated mice as assessed by flow cytometry. However, multiple colon cell types expressed the NMDA-R, suggesting the ability of diverse cells, including CD3-positive cells, CD103-positive cells, Ly6G-positive cells, and epithelial cell adhesion molecule-positive epithelial cells to respond to EI-tPA. Mesenchymal cells that line intestinal crypts and provide barrier function expressed LRP1, thereby representing another potential target for EI-tPA. These results demonstrate that the NMDA-R/LRP1 receptor system may be a target for drug development in diseases characterized by tissue damage and chronic inflammation.

Efficient DNA repair is essential to maintain genomic integrity. An average of 30,000 base lesions per cell are removed daily by the DNA glycosylases of the base excision repair machinery. With the advent of whole genome sequencing, many germline mutations in these DNA glycosylases have been identified and associated with various diseases, including cancer. In this graphical review, we discuss the function of the NTHL1 DNA glycosylase and how genomic mutations and altered function of this protein contributes to cancer and aging. We highlight its role in a rare tumor syndrome, NTHL1-associated polyposis (NAP), and summarize various other polymorphisms in NTHL1 that can induce early hallmarks of cancer, including genomic instability and cellular transformation.

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1β, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.

Abstract The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery, but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell–cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell–cell location. Implications: Rab11-FIP5–dependent α6β1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Mol Cancer Res; 16(8); 1319–31. ©2018 AACR.

Cover: The cover image, by Anne E. Cress., is based on the Article Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells, DOI: 10.1002/jcb.25673.

ABSTRACTLaminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25 min−1, threefold faster than α3 integrin (1.0 min−1), 1.5‐fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71‐fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell–cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8‐fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038–1049, 2017. © 2016 Wiley Periodicals, Inc.

Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non‐myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co‐culture conditions were used to determine if myelinating or non‐myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin‐dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3–2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE‐1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function‐blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non‐myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin‐dependent tumor invasion on laminin. J. Cell. Biochem. 117: 491–499, 2016. © 2015 Wiley Periodicals, Inc.

Abstract Background: Metastatic prostate cancer express laminin binding integrins α6 and α3 while losing the expression of a variety of integrins present in normal prostate gland. Results: In human xenograft tissues, at least 70% of bone and soft tissue metastasis of prostate cancer (185 samples from 45 patients) had cytoplasmic expression of the α6 integrin that correlated to increased expression of intracellular trafficking proteins. Given the reported link between cytoplasmic integrin distribution and integrin endocytosis, surface labelled integrin α6 were quantified for internalization kinetics using flow cytometry. The integrin α6 was internalized 3-fold faster than α3 integrin (CD49c) (1.0 min-1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min-1), and significantly slower than the unrelated transferrin receptor (TfR, CD71) (15 min-1). Silencing α3 expression increased α6 integrin internalization by 1.45 fold without affecting internalization of non-integrin control receptor, TfR. The internalized α6 integrin was targeted to early endosomes and Rab4 recycling vesicles and redistributed to a cell-cell staining pattern similar to human prostate cancer specimens. This was accompanied by a 1.8-fold increase in cancer cell migration that was dependent on α6 integrin. Silencing integrin α6 however, didn't affect the internalization rate or intracellular endocytic distribution of the α3 integrin. Conclusion and Significance: We conclude that internalization of the α6 and α3 integrins is coordinated in a unidirectional fashion and their relative expression can significantly modulate cancer cell migration. The observed loss of α3 integrin expression in high grade cancer is predicted to increase the internalization and pro-migratory features of α6 integrin in high grade cancer. Acknowledgements: Research was supported in part by NIH research grants R01 CA 159406, CA 23074 and CA09213. Patient tissue studies were supported by Biobank and Translational Research Core, Cedars Sinai Medical Center, CA, Pacific Northwest Prostate Cancer SPORE (P50CA97186), the PO1 NIH grants (PO1CA085859) and the Richard M. LUCAS Foundation. Citation Format: Lipsa Das, Todd A. Anderson, Jaime M. Gard, Isis C. Sroka, Stephanie R. Strautman, Raymond B. Nagle, Colm Morrissey, Beatrice S. Knudsen, Anne E. Cress. Loss of α3 integrin expression promotes α6 integrin internalization to Rab4 vesicles and migration of human prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A60.

Abstract Laminin binding integrins, A3B1 and A6B1 drive in part, cancer cell migration and metastasis. Cell migration requires dynamic internalization and recycling of surface integrins. However, whether laminin binding integrin internalization is coordinately or independently controlled is not known. In this study, we quantitated the internalization of integrin A3 and A6 and whether crosstalk influences their respective internalization and subsequent cancer cell migration. We quantitated the internalization of A6 and A3 integrins, using fluorophore conjugated antibody based internalization assays and flow cytometry. In the prostate cancer cell line DU145, we observed constitutive internalization of integrin A6 with a surface half-life of approximately 30 minutes as compared to >70 minutes for integrin A3, 45 minutes for unrelated integrin Av or 10 minutes for the unrelated transferrin receptor. Silencing A3 integrin expression resulted in a 10 percent increase in the total amount of internalized A6 integrin, with a decreased surface half-life of 20 minutes. The internalized A6 integrin showed intracellular punctate staining colocalized with the early endosome marker EEA1 and Rab4 containing vesicles. Increased integrin A6 internalization led to 40% increase in migration which was dependent on A6B1 integrin. Silencing integrin A6 expression, however, did not affect the internalization of A3 integrin indicating a unidirectional regulation of integrin internalization. The changes in the integrin internalization could be clearly ascribed to alpha subunits, as the obtained half-life of A6 and A3 integrins were independent of antibody used or integrin engagement by ligand mimetic peptides. Taken together, these data indicate that A6B1 and A3B1 integrins have different internalization kinetics and the presence of integrin A3 decreased integrin A6 internalization and ensuing cell migration. These data are consistent with previous observations in normal systems suggesting that A3B1 provides a provisional matrix during early stages of wound healing, requiring subsequent stable adhesion complexes and integrin A6 function. In cancer, the loss of A3 integrin and the increased internalization of A6 integrin may account for the “wounds that won't heal” phenotype and provide a strategy for biological intervention to block tumor progression. (Supported in part by NIH Grants CA23074 and CA159406 and the TACMASS core service of the Arizona Cancer Center) Citation Format: Lipsa Das, Todd A. Anderson, Jaime M.c. Gard, Isis C. Sroka, Stephanie R. Strautman, Raymond B. Nagle, Beatrice S. Knudsen, Anne Cress. A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2014-4979

Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.