Citations

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1–expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

ABSTRACT Dynamin is a 100‐kDa GTPase that has been implicated in endocytosis. To extend our understanding of its cellular functions, we have microinjected specific affinity‐purified anti‐dynamin antibodies into cultured mammalian epithelial cells. Using this approach, dynamin function can be inhibited specifically and rapidly in single cells. Effects of microinjected inhibitory antibodies on distinct endocytic processes and plasmalemmal morphology were then assayed by fluorescence microscopy (FM) and ultrastructural analysis. Microinjected antibodies inhibit the clathrin‐mediated endocytosis of fluorophore‐labeled transferrin and cause a marked invagination of the plasma membrane. Many of these long plasmalemmal invaginations had clathrin‐coated pits along their cytoplasmic surface. A number of distinct noncoated pits resembling plasmalemmal caveolae also accumulated in anti‐dynamin antibody‐injected cells. Further, the cellular uptake of cholera toxin B, which normally occurs by the internalization of caveolae, was inhibited in these cells. In support of these observations, immunoisolation techniques, double‐label immuno‐FM, and immunoelectron microscopy (immuno‐EM) provided biochemical and morphological evidence that dynamin associates with plasmalemmal caveolae. Together, these observations indicate that dynamin mediates scission from the plasma membrane of both clathrincoated pits and caveolae during distinct endocytic processes. These results demonstrate that dynamin isoforms are involved in an additional endocytic process that is distinct from clathrinmediated endocytosis and provide significant insights into the molecular mechanisms governing the GTP‐mediated internalization of caveolae. Evidence is provided demonstrating that dynamin isoforms have a differential distribution in mammalian cells. Targeting information for these isoforms is provided at least in part by regions of alternative splicing. Thus, the different dynamin isoforms may be localized to distinct cellular compartments but provide a similar scission function during the biogenesis of nascent cytoplasmic vesicles—Henley, J. R., Cao, H., McNiven, M. A. Participation of dynamin in the biogenesis of cytoplasmic vesicles. FASEB J. 13 (Suppl.), S243‐S247 (1999)

Abstract Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process is dependent upon complex, actin-based specializations of the plasma membrane termed “invadopodia” that act to both sequester and release matrix metalloproteases. Here we report that cells of mesenchymal origin, including fibroblasts, pancreatic stellate cells, and tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. First, as tumor cell-derived invadopodia are dependent upon the action of the GTPase Dyn2, loss of Dyn2 function inhibits invadopodia-based matrix degradation by pancreatic tumor cells. In surprising contrast, we have found that loss of Dyn2 function in mesenchymal cells does not inhibit degradation, but actually leads to a marked increase. Second, these degradative sites lack the punctate shape of conventional invadopodia but are instead spread along the cell base and are reticular and/or fibrous in character. Third, inhibition of either Src or Cdc42 function, known to drive invadopodia formation, had no effect on the mesenchymal matrix degradation induced by loss of Dyn2. Taken together, these data indicate that this mechanism of degradation is independent of classical invadopodial degradation. To define the mechanistic processes that differ between tumor and stromal cell types, we screened for matrix metalloprotease activity using zymography. An unexpected and significant upregulation of MMP2 activity in mesenchymal-derived cells upon loss of Dyn2 was observed, whereas MMP2 was nearly completely absent in the tumor cells. Further, loss of Dyn2 resulted in an upregulation of surface-bound MT1-MMP, known to activate MMP2. In contrast, loss of Dyn2 in the pancreatic tumor cells reduced surface levels of MT1-MMP. Thus, in mesenchymal cells, loss of Dyn2 induces retention of MT1-MMP at the surface, leading to large scale matrix degradation through an MT1-MMP/MMP2-dependent mechanism. Excitingly, co-culture invasion experiments demonstrate that the matrix degradation induced in these stromal fibroblasts is sufficient to promote invasion by pancreatic tumor cells. These findings provide evidence for a novel matrix remodeling process conducted by mesenchymal cells that is substantially more effective than conventional invadopodia, distinct in structural organization, independent of the classical invadopodial machinery, and capable of promoting invasion by pancreatic tumor cells. Supported by NCI RO1 CA104125 to MAM. Citation Format: Hong Cao, Robbin Eppinga, Eugene Krueger, Jing Chen, Gina Razidlo, Mark A. McNiven. Differential mechanisms utilized by pancreatic tumor cells and associated fibroblasts to remodel stroma during metastatic invasion. [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 3162. doi:10.1158/1538-7445.AM2014-3162

Abstract Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5′-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn–Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src–FAK–Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK–Src signaling in turning over FAs.

The size and integrity of the Golgi apparatus is maintained via a tightly controlled regulation of membrane traffic using a variety of different signaling and cytoskeletal proteins. We have recently observed that activation of c-Src has profound effects on Golgi structure, leading to dramatically vesiculated cisternae in a variety of cell types. As the large GTPase dynamin (Dyn2) has been implicated in Golgi vesiculation during secretion, we tested whether inhibiting Dyn2 activity by expression of a Dyn2K44A mutant or siRNA knockdown could attenuate active Src-induced Golgi fragmentation. Indeed, these perturbations attenuated fragmentation, and expression of a Dyn2Y(231/597)F mutant protein that cannot be phosphorylated by Src kinase had a similar effect . Finally, we find that Dyn2 is markedly phosphorylated during the transit of VSV-G protein through the TGN whereas expression of the Dyn2Y(231/597)F mutant significantly reduces exit of the nascent protein from this compartment. These findings demonstrate that activation of Dyn2 by Src kinase regulates Golgi integrity and vesiculation during the secretory process.

Remodeling of cell–cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell–cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell–cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2–binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15–AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.