Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

Advanced prostate cancer (PCa) is known for its high prevalence to metastasize to bone, at which point it is considered incurable. Despite significant effort, there is no animal model capable of recapitulating the complexity of PCa bone metastasis. The humanized mouse model for PCa bone metastasis used in this study aims to provide a platform for the assessment of new drugs by recapitulating the human-human cell interactions relevant for disease development and progression. The humanized tissue-engineered bone construct (hTEBC) was created within NOD-scid IL2rgnull (NSG) mice and was used for the study of experimental PC3-Luc bone metastases. It was confirmed that PC3-Luc cells preferentially grew in the hTEBC compared with murine bone. The translational potential of the humanized mouse model for PCa bone metastasis was evaluated with two clinically approved osteoprotective therapies, the non-species-specific bisphosphonate zoledronic acid (ZA) or the human-specific antibody Denosumab, both targeting Receptor Activator of Nuclear Factor Kappa-Β Ligand. ZA, but not Denosumab, significantly decreased metastases in hTEBCs, but not murine femora. These results highlight the importance of humanized models for the preclinical research on PCa bone metastasis and indicate the potential of the bioengineered mouse model to closely mimic the metastatic cascade of PCa cells to human bone. Eventually, it will enable the development of new effective antimetastatic treatments.

Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here, we focus on understanding cellular mechanisms for elongation using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this locoregional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles that were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the extracellular matrix, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry breaking and elongation. This required β1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating locoregional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.

Women with atypical hyperplasia (AH) or well-differentiated early-stage endometrioid endometrial carcinoma (EEC) who wish to retain fertility and/or with comorbidities precluding surgery, are treated with progestin. Clinically approved predictive biomarkers for progestin therapy remain an unmet need. The objectives of this study were to document the overall response rate (ORR) of levonorgestrel intrauterine device (LNG-IUD) treatment, and determine the association of FGFR2b and FGFR2c expression with treatment outcome. BaseScope RNA ISH assay was utilized to detect expression of FGFR2b and FGFR2c mRNA in the diagnostic biopsies of 89 women (40 AH and 49 EEC) treated with LNG-IUD. Detailed clinical follow-up was available for 69 women which revealed an overall response rate (ORR) of 44% (30/69) with a higher ORR seen in AH (64%) compared to EEC (23%). The recurrence rate in women who initially responded to LNG-IUD was 10/30 (33.3%). RNA ISH was successful in 72 patients and showed FGFR2c expression in 12/72 (16.7%) samples. In the 59 women with detailed clinical follow-up and RNA-ISH data, women with tumours expressing FGFR2c were 5-times more likely to have treatment failure in both univariable (HR 5.08, p < 0.0001) and multivariable (HR 4.5, p < 0.002) Cox regression analyses. In conclusion, FGFR2c expression appears to be strongly associated with progestin treatment failure, albeit the ORR is lower in this cohort than previously reported. Future work to validate these findings in an independent multi-institutional cohort is needed.

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the 'cell cycle' pathway and the 'melanosome'. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and the melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.

Autophagy is a catabolic pathway involving the sequestration of cellular contents into a double-membrane vesicle, the autophagosome. Although recent studies have demonstrated that autophagy supports cell migration, the underlying mechanisms remain unknown. Using live-cell imaging, we uncover that autophagy promotes optimal migratory rate and facilitates the dynamic assembly and disassembly of cell-matrix focal adhesions (FAs), which is essential for efficient motility. Additionally, our studies reveal that autophagosomes associate with FAs primarily during disassembly, suggesting autophagy locally facilitates the destabilization of cell-matrix contact sites. Furthermore, we identify the selective autophagy cargo receptor neighbor of BRCA1 (NBR1) as a key mediator of autophagy-dependent FA remodeling. NBR1 depletion impairs FA turnover and decreases targeting of autophagosomes to FAs, whereas ectopic expression of autophagy-competent, but not autophagy-defective, NBR1 enhances FA disassembly and reduces FA lifetime during migration. Our findings provide mechanistic insight into how autophagy promotes migration by revealing a requirement for NBR1-mediated selective autophagy in enabling FA disassembly in motile cells.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models.

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.

A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Activating FGFR2 mutations have been identified in ~10% of endometrioid endometrial cancers (ECs). We have previously reported that mutations in FGFR2 are associated with shorter disease free survival (DFS) in stage I/II EC patients. Here we sought to validate the prognostic importance of FGFR2 mutations in a large, multi-institutional patient cohort.

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse.

Endometrial carcinoma (EC) is the most common gynecological malignancy and fibroblast growth factor receptor 2 (FGFR2) is a frequently dysregulated receptor tyrosine kinase. FGFR2b and FGFR2c are the two main splice isoforms of FGFR2 and are normally localized in epithelial and mesenchymal cells, respectively. Previously, we demonstrated that FGFR2c mRNA expression was associated with aggressive tumor characteristics, shorter progression-free survival (PFS), and disease-specific survival (DSS) in endometrioid ECs (EECs). The objectives of this study were to investigate the spatial expression of FGFR2b in normal and hyperplasia with and without atypia of human endometrium and to assess the prognostic significance of FGFR2b expression in EC. FGFR2b and FGFR2c mRNA expression was evaluated in normal (proliferative [n = 10], secretory [n = 15], and atrophic [n = 10] endometrium), hyperplasia with and without atypia (n = 19) as well as two patient cohorts of EC samples (discovery [n = 78] and Vancouver [n = 460]) using isoform-specific BaseScope RNA in situ hybridization assays. Tumors were categorized based on FGFR2 isoform expression (one, both, or neither) and categories were correlated with clinicopathologic markers, molecular subtypes, and clinical outcomes. The FGFR2b splice isoform was exclusively expressed in the epithelial compartment of normal endometrium and hyperplasia without atypia. We observed FGFR2c expression at the basalis layer of glands in 33% (3/9) of hyperplasia with atypia. In patients with EEC, FGFR2b+/FGFR2c- expression was found in 48% of the discovery cohort and 35% of the validation Vancouver cohort. In univariate analyses, tumors with FGFR2b+/FGFR2c- expression had longer PFS (hazard ratio [HR] 0.265; 95% CI 0.145-0.423; log-rank p < 0.019) and DSS (HR 0.31; 95% CI 0.149-0.622; log-rank p < 0.001) compared to tumors with FGFR2b-/FGFR2c+ expression in the large EEC Vancouver cohort. In multivariable Cox regression analyses, tumors with FGFR2b+/FGFR2c- expression were significantly associated with longer DSS (HR 0.37; 95% CI 0.153-0.872; log-rank p < 0.023) compared to FGFR2b-/FGFR2c+ tumors. In conclusion, FGFR2b+/FGFR2c- expression is associated with favorable clinicopathologic markers and clinical outcomes suggesting that FGFR2b could play a role in tailoring the management of EEC patients in the clinic if these findings are confirmed in an independent cohort.

Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35-7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09-3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05-0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.

Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.