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The systemic metabolic shifts that occur during aging and the local metabolic alterations of a tumor, its stroma and their communication cooperate to establish a unique tumor microenvironment (TME) that fosters cancer progression. Here, we show that methylmalonic acid (MMA), an aging-increased oncometabolite that is also produced by aggressive cancer cells, activates fibroblasts in the TME, which reciprocally secrete IL-6 loaded extracellular vesicles (EVs) that drive cancer progression, drug resistance and metastasis. The cancer-associated fibroblast (CAF)-released EV cargo is modified as a result of reactive oxygen species (ROS) generation and activation of the canonical and noncanonical TGFβ signaling pathways in CAFs. EV-associated IL-6 functions as a stroma-tumor messenger that activates the JAK/STAT3 and TGFβ signaling pathways in tumor cells and promote an epithelial-to-mesenchymal transition (EMT) and drug resistance in vitro, and metastatic progression in vivo. Our findings reveal the role of MMA in the activation of CAFs to drive metastatic reprogramming, unveiling multiple potential therapeutic avenues to target MMA at the nexus of aging, the tumor microenvironment and metastasis.

The importance of metabolic reprogramming in cancer has been long demonstrated by the association of systemic metabolic changes, such as aging, diet and exercise, with cancer outcomes. These systemic shifts, combined with local metabolic alterations within the tumor microenvironment (TME), can all cooperate to foster an environment conducive to cancer progression. In cells derived from primary and metastatic patient tumors, we found that mesenchymal-like cells displayed dysregulated propionate metabolism, leading to increased accumulation and secretion of methylmalonic acid (MMA), a novel aging-induced oncometabolite. This tumor cell-secreted MMA, in addition to increased MMA in the serum of elderly individuals, combine to form high local accumulation of MMA in the TME. We discovered that MMA acts on fibroblasts in the TME, activating them to cancer-associated fibroblasts (CAFs). MMA modifies the cargo of CAF-secreted extracellular vesicles (EVs), which function as a messenger to tumor cells, further promoting epithelial-to-mesenchymal transition, drug resistance, and increased metastasis in vivo. Here, we reveal a novel function of MMA in cancer, demonstrating for the first time that tumor-secreted MMA recruits the tumor microenvironment to drive cancer progression and metastatic reprogramming. Citation Format: Vivien Low, Zhongchi Li, Ashley Laughney, Wenbing Jin, Noah Dephoure, Valbona Luga, Ethan Earlie, Bobak Parang, Chunjun Guo, Lewis Cantley, John Blenis. Methylmalonic acid in TME signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3175.

Summary Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes) enable neurite growth on CNS inhibitory proteins. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3β and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.

SummaryStroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

SummaryPlanar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. Here, we report an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Further, we show that Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. Our work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE.

Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor–β (TGFβ) pathway. Analysis using self-organizing maps and k -means clustering identified links of the TGFβ pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFβ type I receptor localization for efficient TGFβ-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.

Cancer-associated fibroblasts (CAF) regulate tumor progression, but their role in cancer metastasis remains largely unexplored. Exosomes are secreted microvesicles that are emerging as potent mediators of cell–cell communication that are of particular importance in tumor–stroma interactions. The Wnt-planar cell polarity (PCP) pathway is the primary regulator of convergent extension cell movements during vertebrate development, but the role of this signaling pathway in cancer cell migration and metastasis has been unclear. Recently, we revealed that fibroblasts secrete exosomes that promote breast cancer cell (BCC) protrusive activity, motility, and metastasis by activating autocrine Wnt-PCP signaling in BCCs. Moreover, we showed that Wnt ligands produced by BCCs tether to fibroblast exosomes upon trafficking of exosomes in BCCs. These findings have several implications that motivate promising future research in the fields of tumor–stroma communication, exosome function, and Wnt-PCP signaling in cancer metastasis. Cancer Res; 73(23); 6843–7. ©2013 AACR.

Cell-surface TGFbeta (transforming growth factor beta) receptors partition into membrane rafts and the caveolin-positive endocytic compartment by an unknown mechanism. In the present study, we investigated the determinant in the TGFbeta type II receptor (TbetaRII) that is necessary for membrane raft/caveolar targeting. Using subcellular fractionation and immunofluorescence microscopy techniques, we demonstrated that the extracellular domain of TbetaRII mediates receptor partitioning into raft and caveolin-positive membrane domains. Pharmacological perturbation of glycosylation using tunicamycin or the mutation of Mgat5 [mannosyl(alpha-1,6)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase V] activity interfered with the raft partitioning of TbetaRII. However, this was not due to the glycosylation state of TbetaRII, as a non-glycosylated TbetaRII mutant remained enriched in membrane rafts. This suggested that other cell-surface glycoproteins associate with the extracellular domain of TbetaRII and direct their partitioning in membrane raft domains. To test this we analysed a GMCSF (granulocyte/macrophage colony-stimulating factor)-TbetaRII chimaeric receptor, which contains a glycosylated GMCSF extracellular domain fused to the transmembrane and intracellular domains of TbetaRII. This chimaeric receptor was found to be largely excluded from membrane rafts and caveolin-positive structures. Our results indicate that the extracellular domain of TbetaRII mediates receptor partitioning into membrane rafts and efficient entrance into caveolin-positive endosomes.

Within the tumor microenvironment, cancer-associated fibroblasts (CAFs) are capable of releasing exosomes, small extracellular vesicles, which are then used by breast cancer cells to aid in metastasis. Exosomes are uptaken by the cancer cells and gain Wnt11 through processing, thus allowing them to stimulate Wnt-signaling pathways that causes increased cell motility. Our current research has focused on understanding the events that exosomes undergo as they pass through breast cancer cells. Exosomes from L cells carry the tetraspanin, Cd81, and two of its known transmembrane interactors Igsf8 and Ptgfrn. By using immunoprecipitation, we were able to isolate Cd81-exosomes, and analyze them by mass spectrometry. Furthermore, comparison of Cd81-exosomes before and after processing in MDA-MB-231, a human breast cancer cell line, revealed differences in protein content of the exosomes. Additionally, since L cells are a mouse fibroblast cell line, we could identify the origin of each protein based on variance between human and mouse proteins. Several families of proteins were identified, such as RNA-binding proteins, kinases, phosphatases, and secretory and endocytic proteins. Our data allows us to postulate that Cd81-exosome processing involves the use of the cancer cell endocytic and secretory pathways. By determining the mechanism whereby breast cancer cells use Cd81-exosomes as signals, it may be possible to block this process and prevent or slow metastatic events. Citation Format: Ainsley Q. Underhill, Yingyi Amy Zhang, Liang Zhang, Valbona Luga, Jeff Wrana. Proteomic dynamics of stromal exosomes during processing in cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B62.