Endometrial cancer (EC) is the most commonly diagnosed malignancy of the female reproductive tract. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumors that are refractory to current chemotherapies with an associated poor prognosis. Our laboratory identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid endometrial tumors and have since shown in a large (n=970) multi-institutional cohort they are associated with shorter progression free and cancer specific survival. Although FGFR inhibitors are in clinical trials in several cancer types, no detailed study of the mechanism of cell death has been published. We now show that treatment with BGJ398, AZD4547 and PD173074 leads to the induction of mitochondrial depolarization and changes in metabolic flux in two endometrial cancer cell lines (JHUEM2 and AN3CA) carrying activating mutations (C383R and N550K respectively). Despite this mitochondrial dysfunction, we have convincingly shown that the cell death following FGFR inhibition was caspase-independent, as evidenced by the lack of caspase-3, -7, and -9 activation, absence of PARP cleavage, and the inability of the broad-spectrum caspase inhibitor, Z-VAD-FMK, to prevent cell death. Knockdown of EndoG and AIF, common mediators of caspase-independent death, had no effect. Detailed quantification of LC3 positive puncta shows an increase in autophagy in JHUEM2 and AN3CA cells treated with all FGFR inhibitors. Knockdown of ATG3, ATG7 and ATG12 resulted in a slight increase in Annexin positive cell death indicating that the autophagy was cytoprotective in this context. We have now confirmed this novel caspase-independent cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we have shown that the combination of FGFR inhibitors with the BH3 mimetic ABT737 can markedly augment this caspase-independent cell death which may have implications for the design of more effective clinical trials.

Abstract Endometrial cancer (EC) is the most commonly diagnosed malignancy of the female reproductive tract. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumors that are refractory to current chemotherapies with an associated poor prognosis. Our laboratory identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid endometrial tumors and have since shown in a large (n=970) multi-institutional cohort they are associated with shorter progression free and cancer specific survival. Although FGFR inhibitors are in clinical trials in several cancer types, no detailed study of the mechanism of cell death has been published. We now show that treatment with BGJ398, AZD4547 and PD173074 leads to the induction of mitochondrial depolarization and changes in metabolic flux in two endometrial cancer cell lines (JHUEM2 and AN3CA) carrying activating mutations (C383R and N550K respectively). Despite this mitochondrial dysfunction, we have convincingly shown that the cell death following FGFR inhibition was caspase-independent, as evidenced by the lack of caspase-3, -7, and -9 activation, absence of PARP cleavage, and the inability of the broad-spectrum caspase inhibitor, Z-VAD-FMK, to prevent cell death. Knockdown of EndoG and AIF, common mediators of caspase-independent death, had no effect. Detailed quantification of LC3 positive puncta shows an increase in autophagy in JHUEM2 and AN3CA cells treated with all FGFR inhibitors. Knockdown of ATG3, ATG7 and ATG12 resulted in a slight increase in Annexin positive cell death indicating that the autophagy was cytoprotective in this context. We have now confirmed this novel caspase-independent cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we have shown that the combination of FGFR inhibitors with the BH3 mimetic ABT737 can markedly augment this caspase-independent cell death which may have implications for the design of more effective clinical trials. Citation Format: Leisl Packer, Sara Byron, Samantha Stehbens, Vanessa Bonazzi, David Loch, Andreas Wortmann, Mike Gartside, Nigel Waterhouse, Jennifer Gunter, Pamela M. Pollock. FGFR inhibition in endometrial cancer induces caspase-independent cell death that can be augmented with ABT-737 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B31.

Abstract Preclinical studies by our lab and others have demonstrated Fibroblast Growth Factor Receptor (FGFR) inhibition is a viable therapeutic strategy in FGFR2-mutant endometrial cancer (EC). A significant clinical issue that we aim to address is that of intrinsic and acquired resistance to anti-FGFR therapeutics in the context of EC. Using mutagenesis screens, we have identified mutations in the FGFR2 kinase domain that cause resistance to the FGFR inhibitors dovitinib and NVP-BGJ398 and explore the mechanism of action of the drug-resistant mutations. We previously reported the N550K and V565I mutations caused resistance to dovitinib. The sensitivity/resistance of these FGFR2 mutants to emerging FGFR inhibitors including AZD4547, NVP-BGJ398 and LY2874455 has now been tested. To gain insights into potential mechanisms of acquired resistance to FGFR inhibitors in EC cells we generated subclones of FGFR2mutant AN3CA, MFE280 and JHUEM-2 EC cells resistant to FGFR inhibitors PD173074, Dovitinib and NVP-BGJ398, respectively. Phospho- protein arrays, gene expression profiling, exome sequencing and SNP analysis were performed to identify genes and signalling pathways that mediate resistance to FGFR inhibitors. Sequencing of AN3CAresistant and MFE280resistant subclones excluded secondary FGFR2 mutations as the resistance mechanisms. Upregulation of downstream survival pathways such as pERK and pAKT was observed in some of the JHUEM-2resistant and AN3CAresistant subclones. To further characterise these subclones we are currently analysing data from exome sequencing and copy number analysis, to determine the molecular mechanisms of resistance in these cell lines. In the MFE280resistant cells resistance to FGFR inhibition is associated with an EMT phenotype. Signaling through pERK and pAKT is downregulated in these cells, suggesting that resistance is not mediated by activation of kinase-driven survival pathways. Analysis of expression data is underway to understand how these cells have become resistant to FGFR inhibitors. This research will provide insight into possible combination therapies that may combat drug resistance in FGFR-mutant endometrial cancer. Citation Format: Leisl M. Packer, Sara Byron, David Loch, Farhad Dehkhoda, Andreas Wortmann, Xinyan Geng, Katia Nones, Sean Grimmond, John Pearson, Nic Waddell, Pamela Pollock. Elucidating mechanisms of resistance to FGFR inhibitors in endometrial cancer. [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 1715. doi:10.1158/1538-7445.AM2014-1715

The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and roles in a range of cellular processes, including proliferation, migration, invasion, differentiation, inflammation and angiogenesis that are required in both normal physiology as well as pathological conditions. These roles require cleavage of a range of substrates, including extracellular matrix proteins, growth factors, cytokines as well as other proteinases. In addition, it has been clear since the earliest days of KLK research that cleavage of cell surface substrates is also essential in a range of KLK-mediated cellular processes where these peptidases are essentially acting as agonists and antagonists. In this review we focus on these KLK-regulated cell surface receptor systems including bradykinin receptors, proteinase-activated receptors, as well as the plasminogen activator, ephrins and their receptors, and hepatocyte growth factor/Met receptor systems and other plasma membrane proteins. From this analysis it is clear that in many physiological and pathological settings KLKs have the potential to regulate multiple receptor systems simultaneously; an important issue when these peptidases and substrates are targeted in disease.

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 FGFR2mutant 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 FGFR2mutant 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 FGFR2N550K in JHUEM-2 cells (FGFR2C383R) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Although the dysfunctional activities of proteases in cancer metastasis have been recognised for some time, the particular mechanisms mediating the roles of these enzymes in the spread of cancer remain poorly defined. Recently we have demonstrated that signals initiated by proteolytic cleavage of cell surface receptors can initiate metastasis promoting responses. One of these receptors is the cell surface glycoprotein CUB domain containing protein 1 (CDCP1). Our data indicate that CDCP1 is cleaved at the cell surface in vitro and in vivo by serine proteases thereby initiating intracellular signalling cascades involving activation and docking to CDCP1 of the kinases Src and PKCδ and downstream activation of Akt. Interestingly, we have shown that this cascade protects cancer cells as they disseminate via blood vessels in vivo and we have identified proteases initiating this protection in model systems. Our findings further demonstrate roles for CDCP1 in cell migration in vitro including its competition as a Src substrate with a critical mediator of cell adhesion, FAK, and its role in migration induced by EGFR signalling. Targeting CDCP1 and its signalling effectors may be a rational approach to treat cancer patients at risk of recurrent disease. Support: Wesley Research Institute, Australian Research Council, Cancer Council Queensland, National Health and Medical Research Council of Australia

BACKGROUND: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.RESULTS:The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.CONCLUSIONS:Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCδ to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.

In the last few years dysregulated expression of the cell surface glycoprotein CUB domain‐containing protein 1 (CDCP1) has been associated with several cancers and this cell surface molecule has been recognized both as a tumor marker and as a potential target to disrupt progression of cancer. Here we summarize what is known about CDCP1 including its structural features, expression in normal and cancerous tissues, and the in vitro experiments and studies in animal models that have provided the key insights into its potential role in tumor formation and metastasis in humans. We conclude by highlighting opportunities and challenges in targeting CDCP1 in cancer. © 2009 IUBMB IUBMB Life 61(7): 723–730, 2009

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time- and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2-mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2-mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.