Palmitoylation of the Alternative Amino Terminus of the BTK-C Isoform Controls Subcellular Distribution and Signaling.

BACKGROUND: The alternative transcriptional isoform of Bruton's tyrosine kinase, BTK-C, is expressed in a wide variety of epithelial tumor types where it impacts apoptosis resistance, therapeutic escape, and glucose uptake. The initial exon in BTK-C encodes a 34 amino acid extension of the amino terminus of the canonical BTK-A isoform. Its function is unknown. MATERIALS AND METHODS: Site-directed mutagenesis, acylation assays and expression studies in cancer cell lines were used to determine the effects that the BTK-C first exon sequence has on kinase activity, subcellular localization and cell physiology. Analysis of BTK-C expression in tumors was conducted using genomic databases. RESULTS: BTK-C is palmitoylated on two cysteine residues. BTK-C localization at the plasma membrane is dependent upon phosphatidylinositol 3,4,5-triphosphate (PIP3) levels as well as palmitoylation. In epithelial cancer cells, both BTK-A and BTK-C isoforms are recruited to the plasma membrane; however, BTK-A also localizes to the nucleus whereas BTK-C has a primarily perinuclear distribution. Transcription of the BTK-C isoform is inversely correlated with expression of commonly activated breast cancer signaling receptors in breast tumors. In MDA-MB-231 cells, BTK-C expression confers modest increases in proliferation and glucose uptake rates compared to BTK-A. CONCLUSION: Palmitoylation affects localization and regulation of BTK-C in epithelial tumor cells where it functions as an important survival factor. Expression of either palmitoylated or non-palmitoylated kinase isoforms that function in PI3K signaling may be a common regulatory feature as nine other soluble kinases in the human genome possess similarly encoded alternative N-termini (ANT).

The SHCA adapter protein cooperates with lipoma-preferred partner in the regulation of adhesion dynamics and invadopodia formation.

SHC adaptor protein (SHCA) and lipoma-preferred partner (LPP) mediate transforming growth factor ß (TGFß)-induced breast cancer cell migration and invasion. Reduced expression of either protein diminishes breast cancer lung metastasis, but the reason for this effect is unclear. Here, using total internal reflection fluorescence (TIRF) microscopy, we found that TGFß enhanced the assembly and disassembly rates of paxillin-containing adhesions in an SHCA-dependent manner through the phosphorylation of the specific SHCA tyrosine residues Tyr-239, Tyr-240, and Tyr-313. Using a BioID proximity labeling approach, we show that SHCA exists in a complex with a variety of actin cytoskeletal proteins, including paxillin and LPP. Consistent with a functional interaction between SHCA and LPP, TGFß-induced LPP localization to cellular adhesions depended on SHCA. Once localized to the adhesions, LPP was required for TGFß-induced increases in cell migration and adhesion dynamics. Mutations that impaired LPP localization to adhesions (mLIM1) or impeded interactions with the actin cytoskeleton via α-actinin (ΔABD) abrogated migratory responses to TGFß. Live-cell TIRF microscopy revealed that SHCA clustering at the cell membrane preceded LPP recruitment. We therefore hypothesize that, in the presence of TGFß, SHCA promotes the formation of small, dynamic adhesions by acting as a nucleator of focal complex formation. Finally, we defined a previously unknown function for SHCA in the formation of invadopodia, a process that also required LPP. Our results reveal that SHCA controls the formation and function of adhesions and invadopodia, two key cellular structures required for breast cancer metastasis.

p66ShcA functions as a contextual promoter of breast cancer metastasis.

BACKGROUND: The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. METHODS: We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. RESULTS: We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. CONCLUSION: This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity.

Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of 'illumination overhead' (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.This article has an associated First Person interview with the first author of the paper.

MicroRNA-206 suppresses TGF-ß signalling to limit tumor growth and metastasis in lung adenocarcinoma.

MicroRNA-206 (miR-206) has demonstrated tumor suppressive effects in a variety of cancers. Numerous studies have identified aberrantly expressed targets of miR-206 that contribute to tumor progression and metastasis, however, the broader gene-networks and pathways regulated by miR-206 remain poorly defined. Here, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumors to identify differentially expressed genes, and study the effects on tumor growth and metastasis. In H1299 tumor xenograft assays, stable expression of miR-206 suppressed both tumor growth and metastasis in mice. Profiling of xenograft tumors using small RNA sequencing and a targeted panel of tumor progression and metastasis-related genes revealed a network of genes involved in TGF-ß signalling that were regulated by miR-206. Among these were the TGFB1 ligand, as well as direct transcriptional targets of Smad3. Other differentially expressed genes included components of the extracellular matrix involved in TGF-ß activation and signalling, including Thrombospondin-1, which is responsible for the activation of latent TGF-ß in the stroma. In cultured lung adenocarcinoma cells treated with recombinant TGF-ß, ectopic expression of miR-206 impaired canonical signalling, and expression of TGF-ß target genes linked to epithelial-mesenchymal transition. This was due at least in part to the suppression of Smad3 protein levels in lung adenocarcinoma cells with ectopic miR-206 expression. Together, these findings indicate that miR-206 can suppress tumor progression and metastasis by limiting autocrine production of TGF-ß, and highlight the potential utility of TGF-ß inhibitors for the treatment of lung adenocarcinomas.