Jeremy G.T. Wurtzel

Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

RALBP1/RLIP76 depletion in mice suppresses tumor growth by inhibiting tumor neovascularization

RalBP1/RLIP76 is a widely expressed multifunctional protein that binds the Ral and R-Ras small GTPases. In the mouse, RLIP76 is nonessential but its depletion or blockade promotes tumorigenesis and heightens the sensitivity of normal and tumor cells to radiation and cytotoxic drugs. However, its pathobiologic functions, which support tumorigenesis, are not well understood. Here, we show that RLIP76 is required for angiogenesis and for efficient neovascularization of primary solid tumors. Tumor growth from implanted melanoma or carcinoma cells was blunted in RLIP76(-/-) mice. An X-ray microcomputed tomography-based method to model tumor vascular structures revealed defects in both the extent and form of tumor angiogenesis in RLIP76(-/-) mice. Specifically, tumor vascular volumes were diminished and vessels were fewer in number, shorter, and narrower in RLIP76(-/-) mice than in wild-type mice. Moreover, we found that angiogenesis was blunted in mutant mice in the absence of tumor cells, with endothelial cells isolated from these animals exhibiting defects in migration, proliferation, and cord formation in vitro. Taken together, our results establish that RLIP76 is required for efficient endothelial cell function and angiogenesis in solid tumors.

Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading

In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), whereas R-Ras co-localized with PtdIns(3,4,5)P3 in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P3 at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P3 to ruffles and promoting cell spreading.

RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes.

R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.

Abstract PR09: MicroRNA-24 transferred from platelet-derived microparticles to tumor cells in solid tumors targets mt-Nd2 mRNA and modulates mitochondrial function and tumor growth

The purpose of this study was to evaluate transfer of platelet microRNAs to tumor cells in vivo and to characterize the mechanisms and effects. Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Platelet microparticles in circulating blood have recently been shown to be enriched in platelet-derived microRNAs, and to have the ability to transfer platelet microRNAs to vascular cells as well as some tumor cells when co-incubated in vitro. However, tumor vasculature is highly permeable, allowing the possibility of platelet microparticle-tumor cell interaction. We observed PMP infiltration in the vicinity of blood vessels in grade II/III solid tumors derived from human patients, but not in adjacent normal tissue, including colon carcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, and to a lesser extent in hepatocellular carcinoma; however, we did not observe PMPs in breast invasive ductal carcinoma. To obtain mechanistic insight, we used mouse tumor allograft models, and found that platelet microparticles infiltrated solid tumor allografts, attached to tumor cells, and transferred platelet-derived RNA, including microRNAs, to Lewis lung carcinoma (LLC) tumor cells in vivo, and these effects could be recapitulated by PMP co-incubation in vitro. MiR-24 was not detected in untreated LLC cells but was upregulated following PMP exposure, and in LLC cells isolated from resected tumors. We employed a TU-tagging chemical/genetics approach to selectively tag native platelet RNA, which confirmed transfer of tagged, platelet-derived miR-24 to LLC cells in tumor allografts in vivo. We employed a modified microRNA:mRNA ligation scheme to identify RNA targets of platelet-derived miR-24 in tumor cells, which included mt-Nd2, a mitochondrial mRNA that lacks a 39-UTR typically targeted by microRNAs, and Snora75, a non-coding small nucleolar RNA, thus representing targeting of one non-coding RNA by another. These RNAs were depleted in platelet microparticle-treated cells, resulting in mitochondrial dysfunction and tumor cell growth inhibition, in a miR-24-dependent manner, as these effects of platelet microparticles were rescued with phosphorothioate LNA antagomiR-24. Blockade of miR-24 in tumor cells accelerated tumor growth in vivo. Thus, transfer of platelet microRNAs including miR-24 to tumor cells via microparticles inhibits tumor cell function and tumor progression. However, global depletion of platelet microRNAs by platelet-specific deletion of Dicer1 inhibited tumor angiogenesis, platelet microparticle infiltration, and resulted in delayed allograft tumor growth in mice, indicating a requirement for platelet miRNAs in tumor angiogenesis. These findings add multiple layers to the regulatory roles of platelet-derived microRNAs and platelet microparticles in tumor progression. Citation Format: James V. Michael, Jeremy G.T. Wurtzel, Guang Fen Mao, A. Koneti Rao, Nicholas E. Hoffman, Muniswamy Madesh, Fabian J. Prado, Marvin Nieman, Jesse W. Rowley, Andrew S. Weyrich, Lawrence E. Goldfinger. MicroRNA-24 transferred from platelet-derived microparticles to tumor cells in solid tumors targets mt-Nd2 mRNA and modulates mitochondrial function and tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR09.

Abstract 1870: Inhibition of Galectin-1 sensitizes oncogenic H-Ras to Rapamycin treatment

Specific plasma membrane (PM) localization is essential for H-Ras signaling, and relies on post-translational modifications on the C-terminal targeting domain. H-Ras shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered (Lo/Ld) border upon activation, which is dependent on Galectin-1. We have previously found that H-Ras, which is sequestered in the Lo domain by swapping the C-terminal targeting domain with the Lo-sequestering targeting domain of R-Ras, is deficient in MAPK signal propagation, while having no effect on PI3K activation, nor on H-Ras-driven tumor progression. We have further found that inhibition of PI3K with LY294002 inhibited tumor progression by H-Ras with or without Lo sequestration. Here we show that Lo sequestration of H-Ras attenuated, but did not completely block, H-Ras-induced mTOR signaling (S6kinase phosphorylation) despite similar activation of PI3K as H-Ras. Interestingly, MEK inhibition with U0126 diminished S6kinase phosphorylation by H-Ras, as well as by Lo-sequestered H-Ras. Here we demonstrate that H-Ras-driven allograft tumor growth in mice was substantially blunted upon treatment with mTOR inhibitor Rapamycin, and this effect of Rapamycin was further enhanced in tumors driven by Lo-sequestered H-Ras. Moreover, Rapamycin treatment ablated ERK phosphorylation in H-Ras tumors as well as in tumors with Lo-sequestered H-Ras (in which ERK phosphorylation was already greatly reduced). Together these findings indicate that Lo sequestration of H-Ras inhibits MAPK pathway activation, and that the MAPK pathway engages in crosstalk with mTOR pathways by regulating S6kinase phosphorylation downstream of H-Ras, whereas mTOR activity is required for H-Ras-induced MAPK signaling. These data further indicate that mTOR activation downstream of H-Ras is sufficient to drive tumorigenesis, but this pathway also requires H-Ras-MAPK activation. To recapitulate H-Ras Lo sequestration by the C-terminal targeting domain swap genetic model, we used a Galectin-1 inhibitor, OTX008, to disrupt H-Ras from transitioning between the Lo and Ld domains. OTX008 treatment alone inhibited H-Ras-driven allograft tumor growth to a similar extent as Rapamycin. However, a combination of OTX008 and Rapamycin resulted in nearly complete ablation of H-Ras-dependent tumor growth. These findings indicate that blockade of H-Ras targeting to the lipid ordered/disordered plasma membrane microdomain border, coupled with blockade of mTOR signaling, could provide a novel therapeutic approach to treat H-Ras-associated cancers. Citation Format: James V. Michael, Jeremy G T Wurtzel, Lawrence E. Goldfinger. Inhibition of Galectin-1 sensitizes oncogenic H-Ras to Rapamycin treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1870.

Abstract 2667: Platelet microparticles infiltrating solid tumors transfer miRNAs and modulate tumor angiogenesis and growth

Platelet-derived microparticles are associated with enhancement of metastasis and poor cancer outcomes. Platelet microparticles can transfer platelet microRNAs (miRNAs) to vascular cells upon co-incubation in vitro, but the contributions of platelet microparticles and miRNAs to tumor progression are still poorly understood. Tumor vasculature is highly permeable, allowing the possibility of platelet microparticle-tumor cell interaction in primary solid tumors. Here we show that platelet microparticles infiltrate solid tumors in humans and mice, attach to tumor cells, and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo as well as in vitro. MiR-24 was a major species in this transfer. We identified RNA targets of platelet-derived miR-24 in lung carcinoma cells, which included mt-Nd2, a mitochondrial mRNA which lacks a 3’-UTR, and Snora75, a non-coding small nucleolar RNA. These RNAs were depleted in platelet microparticle-treated tumor cells, resulting in mitochondrial dysfunction and tumor cell growth inhibition, in a miR-24-dependent manner. Blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by platelet microparticle transfusion. Thus, platelet microparticles inhibit lung carcinoma cell proliferation and solid tumor growth via transfer of miR-24. However, global depletion of platelet miRNAs by platelet-specific deletion of Dicer1 inhibited tumor angiogenesis, platelet microparticle infiltration, and delayed tumor growth in mice. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via microparticles, regulate tumor cell gene expression, and modulate tumor progression, whereas platelet miRNAs promote tumor angiogenesis. These findings shed novel insight onto mechanisms of horizontal gene transfer and add multiple layers to the regulatory roles of miRNAs and platelet microparticles in tumor progression. Citation Format: James V. Michael, Jeremy G.T. Wurtzel, Guang Fen Mao, A. Koneti Rao, Nicholas E. Hoffman, Sudarsan Rajan, Muniswamy Madesh, Fabian Jana, Marvin Nieman, Jesse W. Rowley, Andrew S. Weyrich, Lawrence E. Goldfinger. Platelet microparticles infiltrating solid tumors transfer miRNAs and modulate tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2667.

Abstract 2141: Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells

In this study we investigated cellular and molecular mechanisms for breast cancer signaling by EGFR and Ras proteins, independent of activating mutations. Epidermal Growth Factor (EGF) ligation to its receptor, EGFR, at the plasma membrane (PM) activates Ras small GTPases, propagating Ras mitogenic signaling. However, activating Ras mutations are not associated with breast cancers. EGFR and palmitoylated (Pa-) Ras localize to lipid rafts at the PM. Pa-Ras isotypes (H- and N-Ras, but not K-Ras) cycle through vesicle trafficking pathways between the Golgi and PM. We have found that anterograde H-Ras vesicles are sorted in the endocytic recycling compartment (ERC) into vesicles which contain the epithelial-specific transport mediator Rab25 (Rab11c), and Rab11/FIP1C (Rab-coupling protein, RCP). RCP couples Rab25 to vesicles to mediate vesicle anterograde transport. Rab25 and RCP expression are highly correlated with breast cancer progression, aggressiveness, and invasion and metastasis. Rab25, unique among the large family of human Rab genes, has been identified as a major marker and potential stimulator of breast cancers. RCP was recently shown to be a breast cancer driver oncogene, supporting the suggestion that RCP and Rab25 status may be significant clinical markers. Moreover, RCP stimulates H-Ras activation. Meta-analysis of human breast cancer cell line panels shows that while most do not harbor H- or N-Ras mutations, both RCP and Rab25 are highly over-expressed and levels correlate with resistance to EGF and Ras signaling inhibitors. We demonstrate that Rab25, RCP, H-Ras and EGFR co-localized in the ERC and in transport vesicles in MCF10A mammary epithelial cells, and that dominant negative (DN) Rab25 sequestered H-Ras and EGFR from the plasma membrane. DN Rab25 or Rab25 knockdown by shRNA inhibited EGF-induced EGFR phosphorylation, H-Ras activation, and ERK phosphorylation (ppERK), demonstrating a functional link between Rab25 activity and EGFR and H-Ras signaling. Both ER/PR-positive and -negative human breast invasive ductal carcinoma tissues showed elevated ppERK correlated with elevated Rab25 expression over normal breast tissue, whereas HER2+ tumor tissues had elevated ppERK even with low Rab25 expression. Thus, Rab25 and RCP regulate EGFR and Ras trafficking to the plasma membrane, required for mitogenic signaling and associated with tumorigenic effects, even in Ras (WT) cells. Citation Format: Allison B. Herman, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2141. doi:10.1158/1538-7445.AM2015-2141