Matthew D. Buschman

The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.

GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

GOLPH3 is a ubiquitous PI4P effector, critical for Golgi function, and also an oncogene. GOLPH3L, a paralogue in vertebrates, also binds PI4P and localizes to the Golgi, but its expression is restricted to secretory cells. Despite some similarities to GOLPH3, GOLPH3L fails to interact with myosin 18A and functions at the Golgi to antagonize GOLPH3.

GOLPH3 Links the Golgi, DNA Damage, and Cancer

GOLPH3 is the first example of an oncogene that functions in secretory trafficking at the Golgi. The discovery of GOLPH3s roles in both cancer and Golgi trafficking raises questions about how GOLPH3 and the Golgi contribute to cancer. Our recent investigation of the regulation of GOLPH3 revealed a surprising response by the Golgi upon DNA damage that is mediated by DNA-PK and GOLPH3. These results provide new insight into the DNA damage response with important implications for understanding the cellular response to standard cancer therapeutic agents.

The GOLPH3 pathway regulates Golgi shape and function and is activated by DNA damage.

The Golgi protein GOLPH3 binds to PtdIns(4)P and MYO18A, linking the Golgi to the actin cytoskeleton. The GOLPH3 pathway is essential for vesicular trafficking from the Golgi to the plasma membrane. A side effect of GOLPH3-dependent trafficking is to generate the extended ribbon shape of the Golgi. Perturbation of the pathway results in changes to both Golgi morphology and secretion, with functional consequences for the cell. The cellular response to DNA damage provides an example of GOLPH3-mediated regulation of the Golgi. Upon DNA damage, DNA-PK phosphorylation of GOLPH3 increases binding to MYO18A, activating the GOLPH3 pathway, which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival. The PtdIns(4)P/GOLPH3/MYO18A/F-actin pathway provides new insight into the relationship between Golgi morphology and function, and their regulation.

MYO18A: An unusual myosin

MYO18A is a divergent member of the myosin family characterized by the presence of an amino-terminal PDZ domain. MYO18A has been found in a few different complexes involved in intracellular transport processes. MYO18A is found in a complex with LURAP1 and MRCK that functions in retrograde treadmilling of actin. It also has been found in a complex with PAK2, βPIX, and GIT1, functioning to transport that protein complex from focal adhesions to the leading edge. Finally, a high proportion of MYO18A is found in complex with GOLPH3 at the trans Golgi, where it functions to promote vesicle budding for Golgi-to-plasma membrane trafficking. Interestingly, MYO18A has been implicated as a cancer driver, as have other components of the GOLPH3 pathway. It remains uncertain as to whether or not MYO18A has intrinsic motor activity. While many questions remain, MYO18A is a fascinatingly unique myosin that is essential in higher organisms.

The role of Tks adaptor proteins in invadopodia formation, growth and metastasis of melanoma

Metastatic cancer cells are characterized by their ability to degrade and invade through extracellular matrix. We previously showed that the Tks adaptor proteins, Tks4 and Tks5, are required for invadopodia formation and/or function in Src-transformed fibroblasts and a number of human cancer cell types. In this study, we investigated the role of Tks adaptor proteins in melanoma cell invasion and metastasis. Knockdown of either Tks4 or Tks5 in both mouse and human melanoma cell lines resulted in a decreased ability to form invadopodia and degrade extracellular matrix. In addition, Tks-knockdown melanoma cells had decreased proliferation in a 3-dimensional type l collagen matrix, but not in 2-dimensional culture conditions. We also investigated the role of Tks proteins in melanoma progression in vivo using xenografts and experimental metastasis assays. Consistent with our in vitro results, reduction of Tks proteins markedly reduced subcutaneous melanoma growth as well as metastatic growth in the lung. We explored the clinical relevance of Tks protein expression in human melanoma specimens using a tissue microarray. Compared to non-malignant nevi, both Tks proteins were highly expressed in melanoma tissues. Moreover, metastatic melanoma cases showed higher expression of Tks5 than primary melanoma cases. Taken together, these findings suggest the importance of Tks adaptor proteins in melanoma growth and metastasis in vivo, likely via functional invadopodia formation.

Akt/PKB enhances non-canonical Wnt signals by compartmentalizing β-Catenin

Cellular proliferation is antagonistically regulated by canonical and non-canonical Wnt signals; their dysbalance triggers cancers. It is widely believed that the PI3-K→ Akt pathway enhances canonical Wnt signals by affecting transcriptional activity and stability of β-catenin. Here we demonstrate that the PI3-K→Akt pathway also enhances non-canonical Wnt signals by compartmentalizing β-catenin. By phosphorylating the phosphoinositide(PI)-binding domain of a multimodular signal transducer, Daple, Akt abolishes Daple’s ability to bind PI3-P-enriched endosomes that engage dynein motor complex for long-distance trafficking of β-catenin/E-cadherin complexes to pericentriolar recycling endosomes (PCREs). Phosphorylation compartmentalizes Daple/β-catenin/E-cadherin complexes to cell-cell contact sites, enhances non-canonical Wnt signals, and thereby, suppresses colony growth. Dephosphorylation compartmentalizes β-catenin on PCREs, a specialized compartment for prolonged unopposed canonical Wnt signaling, and enhances colony growth. Cancer-associated Daple mutants that are insensitive to Akt mimic a constitutively dephosphorylated state. This work not only identifies Daple as a platform for crosstalk between Akt and the non-canonical Wnt pathway, but also reveals the impact of such crosstalk during cancer initiation and progression.

A Daple-Akt feed-forward loop enhances noncanonical Wnt signals by compartmentalizing β-catenin

Balance between canonical and noncanonical Wnt pathways controls the β-catenin transcriptional program; how the noncanonical pathway antagonizes the canonical pathway remains unclear. We show that Daple, an enhancer of noncanonical Wnt signals, accomplishes that goal by dictating the subcellular distribution of β-catenin in cells.

Abstract 3155: Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Invadopodia are actin-based proteolytic structures which facilitate cancer cell invasion and metastasis. While it was previously believed that invadopodia are not required for tumor cell growth, based on studies in monolayer cell culture, more recently we have noted a role for invadopodia proteins in growth in more physiological 3-dimensional (3D) extracellular matrix contexts. From previous work in our lab, the Tks adaptor proteins with 5 (Tks5) and 4 (Tks4) SH3 domains, respectively, were identified as Src kinase substrates and both are essential molecules for functional invadopodia formation in mouse and human cancer cells. Here, we assess the roles of the Tks adaptors in melanoma tumor growth and invasion both in vivo and in vitro. To address the mechanism as to how the Tks adaptors control tumor growth and invasion, in vitro 3D growth assays in collagen I were performed. Knockdown of either Tks4 or Tks5 in C8161.9 or A375 melanoma cells decreased 3D growth in collagen I, a process that also appeared to be dependent upon metalloproteinase (MMP) activity. Interestingly, knockdown of either Tks4 or Tks5 in A375 melanoma cells decreased cell-surface expression of MT1-MMP, a key protease involved in tumor growth and metastasis. In vivo tumor growth and metastasis assays in mice using human and mouse melanoma cells depleted of Tks4 or Tks5 by shRNA revealed roles for the Tks adaptors in both the size and the number of lung metastases. Finally, analysis of a human melanoma tissue array with a Tks5 antibody, and RNAseq data, indicates that Tks5 expression is increased in lymph node and other metastases in comparison to nevi and primary sites. These data suggest that the Tks adaptors control melanoma tumor growth and invasion by regulating invadopodia formation and perhaps also cell surface expression of MT1-MMP, and thus contribute to melanoma progression. Citation Format: Shinji Iizuka, Christine M. Gould, Matthew D. Buschman, Diaz Begona, Christopher Abdullah, Sara Courtneidge. Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma. [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 3155. doi:10.1158/1538-7445.AM2014-3155