Identification of acquired Notch3 dependency in metastatic Head and Neck Cancer.

During cancer development, tumor cells acquire changes that enable them to invade surrounding tissues and seed metastasis at distant sites. These changes contribute to the aggressiveness of metastatic cancer and interfere with success of therapy. Our comprehensive analysis of "matched" pairs of HNSCC lines derived from primary tumors and corresponding metastatic sites identified several components of Notch3 signaling that are differentially expressed and/or altered in metastatic lines and confer a dependency on this pathway. These components were also shown to be differentially expressed between early and late stages of tumors in a TMA constructed from over 200 HNSCC patients. Finally, we show that suppression of Notch3 improves survival in mice in both subcutaneous and orthotopic models of metastatic HNSCC. Novel treatments targeting components of this pathway may prove effective in targeting metastatic HNSCC cells alone or in combination with conventional therapies.

High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFß and hippo effectors in mammalian cells.

Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFß/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level. For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020).

ROCK inhibitors upregulate the neuroprotective Parkin-mediated mitophagy pathway.

The accumulation of damaged mitochondria causes the death of dopaminergic neurons. The Parkin-mediated mitophagy pathway functions to remove these mitochondria from cells. Targeting this pathway represents a therapeutic strategy for several neurodegenerative diseases, most notably Parkinson's disease. We describe a discovery pipeline to identify small molecules that increase Parkin recruitment to damaged mitochondria and ensuing mitophagy. We show that ROCK inhibitors promote the activity of this pathway by increasing the recruitment of HK2, a positive regulator of Parkin, to mitochondria. This leads to the increased targeting of mitochondria to lysosomes and removal of damaged mitochondria from cells. Furthermore, ROCK inhibitors demonstrate neuroprotective effects in flies subjected to paraquat, a parkinsonian toxin that induces mitochondrial damage. Importantly, parkin and rok are required for these effects, revealing a signaling axis which controls Parkin-mediated mitophagy that may be exploited for the development of Parkinson's disease therapeutics.

Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models.

The alkylating agent platinum is first-line chemotherapy treatment for high-grade serous carcinomas (HGSC) of tubal-ovarian origin. Platinum compounds cause DNA damage and induce apoptotic cell death in the bulk tumor population. However, subpopulations of tumor cells may exhibit diverging behaviors from the bulk tumor due to an alternate stress response that diverts tumor cells from apoptotic death. In this study, we identified a salvage survival pathway in which G2-arrested tumor cells bypassed apoptosis and progressed through aberrant mitotic events to then emerge as a distinct subpopulation of viable large hyperploid cells but with uncertain long-term propagation potential. Platinum-induced large hyperploid cells were flow sorted and showed rare regrowth capacity as compared to their more proficiently regenerating non-hyperploid counterparts. However, detailed time-lapse microscopy provided direct evidence that these hyperploid cells were mitotically active and could divide successfully to produce viable daughter cells. The hyperploid survival response was observed across different cell lines and utilization of this survival pathway was dependent on the strength of the G2-M checkpoint. Conceivably, this salvage survival strategy may contribute to increased genomic diversity of the regenerating tumor cell line through a coupled hyperploidization and de-polyploidization process that may be relevant for drug resistance.

Atypical function of a centrosomal module in WNT signalling drives contextual cancer cell motility.

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.

PTEN regulates cilia through Dishevelled.

Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies.

CEP192 interacts physically and functionally with the K63-deubiquitinase CYLD to promote mitotic spindle assembly.

CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans. Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192's function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape. To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis. Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.

Extracellular phosphorylation of collagen XVII by ecto-casein kinase 2 inhibits ectodomain shedding.

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-alpha converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [gamma-(32)P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser(542) and Ser(544) in the ectodomain as phosphate group acceptors. Phosphorylation of Ser(544) in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitope-specific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser(544) to Ala significantly increased ectodomain shedding, whereas overexpression of CK2alpha inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.

Ciliary calcium signaling is modulated by kidney injury molecule-1 (Kim1).

Primary cilia have been shown to play an important role in embryonic development as well as in postnatal life. Dysfunctional cilia are associated with situs inversus, retinal abnormalities, impaired mucociliary clearance, infertility, hydrocephalus, and congenital renal cysts. In autosomal dominant polycystic kidney disease, mutations of the ciliary proteins polycystin1 or the transient receptor potential (TRP) channel family protein polycystin2 (TRPP2) cause progressive cyst formation and destruction of the kidney. Primary cilia act as flow sensors and respond to flow-mediated bending with a prolonged intracellular calcium increase, which appears to require an intact polycystin protein complex. We have established a novel flow chamber system, which allows us to study renal epithelial cells by live cell imaging. We show that MDCK cells respond to flow by a delayed increase in intracellular calcium and that this response requires these cells to be ciliated. We show that a novel interactor of TRPP2, kidney injury molecule-1 (Kim1), which is expressed at low levels in the normal kidney and upregulated after ischemia, in renal cell cancer and in PKD is targeted to primary cilia when stably expressed in MDCK cells. We demonstrate that expression of tyrosine mutant Kim1, lacking a conserved tyrosine in the intracellular tail, abolishes the calcium increase in response to flow in a dominant negative manner. These results establish Kim1 as a novel regulatory molecule of flow-induced calcium signaling.

Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways.

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.