Collagen fibril formation at the plasma membrane occurs independently from collagen secretion.

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

UK-wide major trauma center tertiary trauma survey pro forma review and aggregation and consolidation into a redesigned document.

Objectives: The trauma tertiary survey (TTS) is an essential part of the continued care for major trauma patients which is performed to ensure that all injuries have been identified and none have been overlooked during the patient's stay. Although the Advanced Trauma Life Support Course states a need for a tertiary survey, there is currently no standard for what this survey comprises. Methods: Using local consultant expert opinion and a literature search we identified a set of 32 TTS potential features that may be included within a TTS pro forma. Major trauma center (MTC) documents were requested from every MTC within the UK. 4 investigators sequentially interrogated each MTC TTS document looking for (1) presence of each feature and (2) how well the feature was represented on the document (0 to 4 Likert Scale). Any previously unidentified potential TTS features were noted and later reviewed for a second round of document analysis. Results: A total of 21 out of all 26 UK MTCs had a TTS pro forma document. A total of 68 possible features were identified. Respiratory and Abdominal assessment sections were the most frequently identified features (present in 90.4% of the TTS pro formas; n=19. Neck assessment and neurological assessment were included within 85.7% of the TTS pro formas (n=18). Further aspects identified for Round 2 analysis typically included features that were thought to be important but highly specific. For example, pregnancy test and DNACPR discussions were found in 1 MTC TTS each (4%). Conclusion: This article presents a review of the existing documents at 21 MTCs in the UK, identification of features used and proposes a gold standard TTS which can be used by any doctor to perform the tertiary survey and reduce the risk of missed injuries in trauma patients. Level of Evidence: 3.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten-/-/trp53-/- mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-ß, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

Circadian control of the secretory pathway maintains collagen homeostasis.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

Preservation of circadian rhythms by the protein folding chaperone, BiP.

Dysregulation of collagen synthesis is associated with disease progression in cancer and fibrosis. Collagen synthesis is coordinated with the circadian clock, which in cancer cells is, curiously, deregulated by endoplasmic reticulum (ER) stress. We hypothesized interplay between circadian rhythm, collagen synthesis, and ER stress in normal cells. Here we show that fibroblasts with ER stress lack circadian rhythms in gene expression upon clock-synchronizing time cues. Overexpression of binding immunoglobulin protein (BiP) or treatment with chemical chaperones strengthens the oscillation amplitude of circadian rhythms. The significance of these findings was explored in tendon, where we showed that BiP expression is ramped preemptively prior to a surge in collagen synthesis at night, thereby preventing protein misfolding and ER stress. In turn, this forestalls activation of the unfolded protein response in order for circadian rhythms to be maintained. Thus, targeting ER stress could be used to modulate circadian rhythm and restore collagen homeostasis in disease.-Pickard, A., Chang, J., Alachkar, N., Calverley, B., Garva, R., Arvan, P., Meng, Q.-J., Kadler, K. E. Preservation of circadian rhythms by the protein folding chaperone, BiP.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.

ΔNp63γ/SRC/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers.

Purpose: To investigate the regulation of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and its importance in tumor invasion.Experimental Design: We use a three-dimensional invasive organotypic raft culture model of human foreskin keratinocytes expressing the E6/E7 genes of the human papilloma virus-16, coupled with bioinformatic and IHC analysis of patient samples to investigate the role played by EMT in invasion and identify effectors and upstream regulatory pathways.Results: We identify SNAI2 (Slug) as a critical effector of EMT-activated downstream of TP63 overexpression in HNSCC. Splice-form-specific depletion and rescue experiments further identify the ΔNp63γ isoform as both necessary and sufficient to activate the SRC signaling axis and SNAI2-mediated EMT and invasion. Moreover, elevated SRC levels are associated with poor outcome in patients with HNSCC in The Cancer Genome Atlas dataset. Importantly, the effects on EMT and invasions and SNAI2 expression can be reversed by genetic or pharmacologic inhibition of SRC.Conclusions: Overexpression of ΔNp63γ modulates cell invasion by inducing targetable SRC-Slug-evoked EMT in HNSCC, which can be reversed by inhibitors of the SRC signaling. Clin Cancer Res; 24(16); 3917-27. ©2018 AACR.

The IGF axis in HPV associated cancers.

Human papillomaviruses (HPV) infect and replicate in stratified epithelium at cutaneous and mucosal surfaces. The proliferation and maintenance of keratinocytes, the cells which make up this epithelium, are controlled by a number of growth factor receptors such as the keratinocyte growth factor receptor (KGFR, also called fibroblast growth factor receptor 2b (FGFR2b)), the epithelial growth factor receptor (EGFR) and the insulin-like growth factor receptors 1 and 2 (IGF1R and IGF2R). In this review, we will delineate the mutation, gene transcription, translation and processing of the IGF axis within HPV associated cancers. The IGFs are key for developmental and postnatal growth of almost all tissues; we explore whether this crucial axis has been hijacked by HPV.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling.

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures.

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner. In this mini-review, we discuss the factors influencing the variation in IGFBP2 expression in cancer and our interpretation of these findings.

AKT in stromal fibroblasts controls invasion of epithelial cells.

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner. By depleting stromal fibroblasts of each of the three AKT isoforms independently, or through using isoform specific inhibitors, we determined that stromal AKT2 is an essential regulator of invasion and show in oro-pharyngeal cancers that AKT2 specific phosphorylation events are also identified in stromal fibroblasts. Depletion of stromal AKT2 inhibits epithelial invasion through activating a protective pathway counteracting KGF mediated invasions. AKT2 depletion in fibroblasts stimulates the cleavage and release of IL1B from stromal fibroblasts resulting in down-regulation of the KGF receptor (fibroblast growth factor receptor 2B (FGFR2B)) expression in the epithelium. We also show that high IL1B is associated with increased overall survival in a cohort of patients with oro-pharyngeal cancers. Our findings demonstrate the importance of stromal derived growth factors and cytokines in regulating the process of tumour cell invasion.

Regulation of epithelial differentiation and proliferation by the stroma: a role for the retinoblastoma protein.

Signaling between the epithelium and stromal cells is crucial for growth, differentiation, and repair of the epithelium. Although the retinoblastoma protein (Rb) is known to regulate the growth of keratinocytes in a cell-autonomous manner, here we describe a function of Rb in the stromal compartment. We find that Rb depletion in fibroblasts leads to inhibition of differentiation and enhanced proliferation of the epithelium. Analysis of conditioned medium identified that keratinocyte growth factor (KGF) levels were elevated following Rb depletion. These findings were also observed with organotypic co-cultures. Treatment of keratinocytes with KGF inhibited differentiation and enhanced keratinocyte proliferation, whereas reduction of KGF levels in Rb-depleted fibroblasts was able to restore expression of differentiation markers. Our findings suggest a crucial role for dermal fibroblasts in regulating the differentiation and proliferation of keratinocytes, and we demonstrate a role for stromal Rb in this cross-talk.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.