Perineural invasion (PNI) is an ominous event strongly linked to poor clinical outcome. Cells residing within peripheral nerves collaborate with cancer cells to enable PNI, but the contributing ...conditions within the tumor microenvironment are not well understood. Here, we show that CCR2-expressing inflammatory monocytes (IM) are preferentially recruited to sites of PNI, where they differentiate into macrophages and potentiate nerve invasion through a cathepsin B-mediated process. A series of adoptive transfer experiments with genetically engineered donors and recipients demonstrated that IM recruitment to nerves was driven by CCL2 released from Schwann cells at the site of PNI, but not CCL7, an alternate ligand for CCR2. Interruption of either CCL2-CCR2 signaling or cathepsin B function significantly impaired PNI
Correlative studies in human specimens demonstrated that cathepsin B-producing macrophages were enriched in invaded nerves, which was associated with increased local tumor recurrence. These findings deepen our understanding of PNI pathogenesis and illuminate how PNI is driven in part by corruption of a nerve repair program. Further, they support the exploration of inhibiting IM recruitment and function as a targeted therapy for PNI.
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The ability of cancer cells to invade along nerves is associated with aggressive disease and diminished patient survival rates. Perineural invasion (PNI) may be mediated by nerve secretion of glial ...cell line-derived neurotrophic factor (GDNF) attracting cancer cell migration through activation of cell surface Ret proto-oncogene (RET) receptors. GDNF family receptor (GFR)α1 acts as coreceptor with RET, with both required for response to GDNF. We demonstrate that GFRα1 released by nerves enhances PNI, even in the absence of cancer cell GFRα1 expression. Cancer cell migration toward GDNF, RET phosphorylation, and MAPK pathway activity are increased with exposure to soluble GFRα1 in a dose-dependent fashion. Dorsal root ganglia (DRG) release soluble GFRα1, which potentiates RET activation and cancer cell migration. In vitro DRG coculture assays of PNI show diminished PNI with DRG from GFRα1 ⁺/⁻ mice compared with GFRα1 ⁺/⁺ mice. An in vivo murine model of PNI demonstrates that cancer cells lacking GFRα1 maintain an ability to invade nerves and impair nerve function, whereas those lacking RET lose this ability. A tissue microarray of human pancreatic ductal adenocarcinomas demonstrates wide variance of cancer cell GFRα1 expression, suggesting an alternate source of GFRα1 in PNI. These findings collectively demonstrate that GFRα1 released by nerves enhances PNI through GDNF-RET signaling and that GFRα1 expression by cancer cells enhances but is not required for PNI. These results advance a mechanistic understanding of PNI and implicate the nerve itself as a key facilitator of this adverse cancer cell behavior.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting ...the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.
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► AP-1A sorts cargo proteins from the TGN to the basolateral plasma membrane ► Loss of AP-1A causes cargo proteins to spill over into recycling endosomes ► AP-1B sorting function at recycling endosomes compensates for AP-1A knockdown ► Transferrin receptor basolateral signal binds noncanonical sites in AP-1A and AP-1B
Gravotta et al. find that the ubiquitous clathrin adaptor AP-1A sorts cargo proteins from the trans-Golgi network to the basolateral membrane. AP-1A knockdown reroutes cargo into recycling endosomes for compensatory sorting by the epithelial-specific adaptor AP-1B. AP-1A and AP-1B are thus partially redundant, mediating basolateral sorting via distinct routes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Perineural invasion (PNI) is an ominous form of cancer progression along nerves associated with poor clinical outcome. Glial derived neurotrophic factor (GDNF) interacts with cancer cell RET ...receptors to enable PNI, but downstream events remain undefined. We demonstrate that GDNF leads to early activation of the GTPase Cdc42 in pancreatic cancer cells, but only delayed activation of RhoA and does not affect Rac1. Depletion of Cdc42 impairs pancreatic cancer cell chemotaxis toward GDNF and nerves. An siRNA library of guanine nucleotide exchange factors was screened to identify activators of Cdc42. ARHGEF7 (β-Pix) was required for Cdc42 activation and chemotaxis toward nerves, and also colocalizes with RET under GDNF stimulation. Cdc42 enables PNI in an
dorsal root ganglia coculture model, and controls the directionality of migration but does not affect cell speed or cell viability. In contrast, Rac1 was necessary for cell speed but not directionality, while the RhoA was not necessary for either cell speed or directionality. Cdc42 was required for PNI in an
murine sciatic nerve model. Depletion of Cdc42 significantly diminished the length of PNI, volume of PNI, and motor nerve paralysis resulting from PNI. Activated Cdc42 is expressed in human salivary ductal cancer cells invading nerves. These findings establish the GDNF-RET-β-Pix-Cdc42 pathway as a directional regulator of pancreatic cancer cell migration toward nerves, highlight the importance of directional migration in PNI, and offer novel targets for therapy. IMPLICATIONS: Cdc42 regulates cancer cell directional migration toward and along nerves in PNI.
Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular ...signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective ...cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Perineural invasion is a form of cancer progression where cancer cells invade along nerves. This behavior is associated with poor clinical outcomes; therefore, it is critical to identify novel ...ligand-receptor interactions between nerves and cancer cells that support the process of perineural invasion. A proteomic profiler chemokine array was used to screen for nerve-derived factors secreted from tissue explants of dorsal root ganglion (DRG), and CCL2 was identified as a lead candidate. Prostate cancer cell line expression of CCR2, the receptor to CCL2, correlated closely with MAPK and Akt pathway activity and cell migration towards CCL2 and DRG. In vitro nerve and cancer coculture invasion assays of perineural invasion demonstrated that cancer cell CCR2 expression facilitates perineural invasion. Perineural invasion is significantly diminished in coculture assays when using DRG harvested from CCL2(-/-) knockout mice as compared with control CCL2(+/+) mice, indicating that CCR2 is required for perineural invasion in this murine model of perineural invasion. Furthermore, 20 of 21 (95%) patient specimens of prostate adenocarcinoma with perineural invasion exhibited CCR2 expression by immunohistochemistry, while just 3 of 13 (23%) lacking perineural invasion expressed CCR2. In summary, nerve-released CCL2 supports prostate cancer migration and perineural invasion though CCR2-mediated signaling.
These results reveal CCL2-CCR2 signaling as a key ligand-receptor mechanism that mediates cancer cell communication with nerves during perineural invasion and highlight a potential future therapeutic target.
Recent advances in cancer neuroscience necessitate the systematic analysis of neural influences in cancer as potential therapeutic targets in oncology. Here, we outline recommendations for future ...preclinical and translational research in this field.
Recent studies have demonstrated a critical role for nerves in enabling tumor progression. The association of nerves with cancer cells is well established for a variety of malignant tumors, including ...pancreatic, prostate and the head and neck cancers. This association is often correlated with poor prognosis. A strong partnership between cancer cells and nerve cells leads to both cancer progression and expansion of the nerve network. This relationship is supported by molecular pathways related to nerve growth and repair. Peripheral nerves form complex tumor microenvironments, which are made of several cell types including Schwann cells. Recent studies have revealed that Schwann cells enable cancer progression by adopting a de-differentiated phenotype, similar to the Schwann cell response to nerve trauma. A detailed understanding of the molecular and cellular mechanisms involved in the regulation of cancer progression by the nerves is essential to design strategies to inhibit tumor progression.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Nerves enable cancer progression, as cancers have been shown to extend along nerves through the process of perineural invasion, which carries a poor prognosis. Furthermore, the innervation of some ...cancers promotes growth and metastases. It remains unclear, however, how nerves mechanistically contribute to cancer progression. Here, we demonstrated that Schwann cells promote cancer invasion through direct cancer cell contact. Histological evaluation of murine and human cancer specimens with perineural invasion uncovered a subpopulation of Schwann cells that associates with cancer cells. Coculture of cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner. Upon contact, Schwann cells induced the formation of cancer cell protrusions in their direction and intercalated between the cancer cells, leading to cancer cell dispersion. The formation of these processes was dependent on Schwann cell expression of neural cell adhesion molecule 1 (NCAM1) and ultimately promoted perineural invasion. Moreover, NCAM1-deficient mice showed decreased neural invasion and less paralysis. Such Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead exploited by cancer cells to promote perineural invasion and cancer progression.
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