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Introduction. Endomucin-1 and endomucin-2 are two proteins structurally related, yet with low homology. The membrane bound sialoglycoproteins appear to play a key role in interfering with the formation of focal adhesion complexes (FAC) and matrix adhesiveness of cells, by mechanisms independent of the MUC1 repeat, which the endomucins do not possess. Endomucins are thought to be expressed at high levels in endothelium and haematopietic cell lineages, although the levels in mammary tissues also seem high. Despite the seeming importance of endomucins in the adhesion and migration of cells, including cancer cells, the clinical value of endomucin in clinical cancer, including breast cancer, is largely unknown. The present study examined the expression profile of endomucins, together with the focal adhesion kinase FAK, in breast cancer and aimed to explore the cellular impact of endomucin on cancer cells. Methods. Human breast cancer cells MCF-7 and MDA MB-231 and a range of other cell types were used. An endomucin overexpression cell model was created and subsequently used to evaluate the function of the cells. The expression profile of the endomucin-1 and endomucin-2 transcripts and FAK, in an existing fresh frozen breast cancer tissue cohort, were quantified. Results. High levels of endomucins, particularly endomucin-1, are good indicators for the overall survival of the patient, p=0.021 for endomucin-1 and p=0.15 for endomucin-2. When expression levels of FAK were integrated into the survival analysis model, patients with high levels of both endomucins and low levels of FAK had the most favourable outcome, compared with those with most unfavourable outcome who had low level of endomucin and high FAK (survival during the follow up period respectively at 100% and 54%, p=0.013, Harzoud Ratio 0.298). Together with the Nottingham Prognostic Index, which independently predicts a poor outcome (p=0.009, HR=7.6), the integrated expression profile of endomucin/FAK represents an independent prognostic indicator for favourable overall survival (p=0.003, HR=0.13), and indeed for a favourable disease free survival (p=0.008, HR=0.17). Mammary tissues, and indeed breast cancer cell lines, expressed high levels of endomucin-2 transcripts and low levels of endomucin-1 transcripts. High levels of endomucin-2 were also seen in fibroblasts and vascular endothelial cells. We created a breast cancer cell submodel with MCF-7, by overexpressing endomucin. It was shown that although endomucin over-expression had some marginal impact on the adhesiveness of breast cancer cells, the over-expression however, had significant impact on cells’ sensitivity to FAK inhibitor, with a markedly reduced adhesiveness to matrix (p< 0.001 control versus endomucin overexpression cells). Discussion. Endomucins have a reduced expression in breast cancers and the reduction, together with low levels of focal adhesion kinase, facilitate a favourable outcome for the patients. Together with the findings of in vitro cell models, it would suggest that the expression profile of endomucins and FAK may be a good indicator, not only for evaluating clinical outcomes, but also for choice of target therapies. Citation Format: Amber Xinyu Li, Tracey A. Martin, Lin Ye, Andrew J. Sanders, Fiona Ruge, Jane Lane, Eleri Davies, Wen G. Jiang. Endomucins and their expression in breast cancer and the cellular and therapeutic impact [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-17.

Introduction. Claudin-10 (CLDN10), a member of the Claudin tight junction (TJ) protein family, was initially known as Oligodendrocyte-Specific Protein-Like (OSP-L). The distribution of CLDN10 in the body is not ubiquitous and is seen more abundantly in kidney and brain and at modest levels in mammary tissues. Limited information suggests that in cells where CLDN10 is expressed, it interacts with other family members including CLDN-11, 12, 16, 17 and the subcoat zonula occludens (ZO-1 and ZO-3) to regulate TJ functions. The role played by CLDN10 in cancer is not clear but there are suggestions that it is a favourable indicator for disease progression in kidney and gastric cancers but an adverse indictor in hormone related cancers such as thyroid cancer. The role of CLDN10 in breast cancer is otherwise not known. We have investigated the expression profile and in particular in its relationship with hormone receptor status. Methods. We carried out CLDN10 transcript and protein analyses by way of quantitative PCR and immunohistochemistry on an established cohort of fresh frozen mammary tissues and breast cancer tissues. Expression was analysed via QPCR against the staging, histology, clinical outcome, hormone status including ER and Her family members, and also its known interactive TJ molecules available to our database. Results. Compared with its interactive TL partner molecules, CLDN10 was expressed at relatively low levels in mammary tissues. There was no significant difference in CLDN10 transcript levels between normal tissues and tumour tissues. Patients with high levels of CLDN10 in breast tumours had significantly shorter overall survival (OS) (p=0.041) and disease free survival (DFS) (p=0.026). In our cohort, we found a significant correlation between levels of CLDN10 and Her-1/EGFR (r=0.201, p=0.026) and Her3 (r=0.935, p< 0.0001), but not with Her-2 nor with Her-4. It was also surprised to find that CLDN10 did not correlated with the ZO-1, ZO-3, CLDN11, 12, 14, and 17. We then identified a subgroup of patients with low levels of CLDN10 and their overall survival (OS) and disease free survival (DFS) which were significantly correlated with Her-1/Her-3 expression status (p< 0.0001 for both OS and DFS). CLDN10, amongst the clinical, pathological and hormone receptor status, is an independent prognostic factor for DFS (p=0.008, Hazard Ratio (HR) =4.228 (95% CI 1.449-12.342)) and for OS (p=0.025, HR=3.917 (95% CI 1.187-12.924)). The predictive power for triple negative breast cancer (TNBC) and non-TNBC by CLDN10 otherwise remained similar and not statistically significant. It was noted that stratification by ER and Her-2 did not contribute to the value of independency. Conclusion. CLDN10, although expressed at relative lower levels in breast cancer, is a contributing factor to the survival of the patients. Together with the status of EGFR and Her-3, it makes an independent prognostic factor for both the overall survival and disease free survival of the patients. Citation Format: Xinguo Zhuang, Wen G. Jiang, Fiona Ruge, Eleri Davies, Bing Xu, Tracey A. Martin. The Expression of Claudin-10 in relationship with Her family members in patients with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-29.

A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer Han Gao1, Fiona Ruge1, Lin Ye1, Jane Lane1, Eleri Davies2, Wen G. Jiang1, Tracey A. Martin1 1Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK 2Wales Breast Centre, University Llandough Hospital, Cardiff CF64 2XX, UK Introduction. MUPP1 (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) or MPDZ is a scaffold protein with multiple PDZ domains, critical for multiple protein docking and protein-protein interactions in cells. MUPP1 is particularly interesting as it offers docking sites for a number of tight junction (TJ) proteins and proteins known to regulate cell junction and motility, including angiomotin (AMOT) and angiomotin-like proteins, the latter being important in regulating cell migration of endothelial cells and an important member in TJ protein complexes. The MUPP1 protein complex at cell junctions also includes CAR/CXADR (coxsackie adenovirus receptor) and members of the ERM (Moesin-Ezrin-Radixin) protein family. It was proposed that the junctional-related MUPP1 docking protein complex may collectively have a role in the development and progression of cancer including breast cancer. The present study examined the profile and integrated expression of these MUPP1 docking molecules in relationship with the outcome, clinical and pathological factors of breast cancer. Methods. Using the datasets of an existing breast cancer cohort at Cardiff, we visited the expression profile of the MUPP1/MPDZ docking molecules including MUPP1, four members of the ERM family, including ezrin, moesin, radixin and EHM2 (also known as NF2) and CAR/CXADR (coxsackie adenovirus receptor). The profile and integrated expression of the profile were analysed against clinical, pathological and outcome factors and also the hormone receptor status of breast cancers. Results. MUPP1 is a weakly linked to the disease progression, so as angiomotin, angiomotin-like1 and angiomotin-like2. Alone, only EHM2 showed significance in linking with the survival of the patient’s OS (p=0.38), neither of the remaining MUPP1 complex molecules, including MUPP1, AMOT, AMOTL1, AMOTL2, ezrin, moesin and radixin was able to offer a significant predictive value for the survival of the patients. However, it was found that MUPP1 has a significant correlation with AMOTs, EHM2 and ezrin. When these significantly correlated molecules were integrated in the analysis, the integrated expression pattern showed a highly significant value in evaluating the overall survival (p=0.003) and disease-free survival (p=0.007). Both of these predictive powers are independent in a multivariate analyses including the clinical and hormone receptors for OS (p=0.022, Hazard Ratio (HR)=1.352 95%CI (1.044-1.752)) and RFS (p=0.023, HR=1.241 (95% CI. 1.031-1.498)). It was interesting to note that the link between the expression pattern and OS was more prominent in ER negative (p=0.018) than in ER positive tumours (p=0.177). The same was observed with disease free survival for ER negative (p=0.011) compared with ER positive (p=0.462) tumours. Non-triple negative breast cancer had shown a similar sensitivity compared with triple negative breast cancer. Discussion. MUPP1, a PDZ domain containing docking protein closed linked with TJ structure, forms an interesting expression profile with its docking molecules including angiomotins and EHM2 that allows evaluation of disease progress and clinical outcome of the patients with breast cancer. Citation Format: Han Gao, Fiona Ruge, Lin Ye, Jane Lane, Eleri Davies, Wen G. Jiang, Tracey A. Martin. A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-35.

Introduction. The striatin protein family are calmodulin binding scaffolding proteins. Striatins have been involved in cell signalling and recently found also acting as cell adhesion molecules and adhesion regulators in adherens junctions and tight junctions. Straitins, particularly striatin-2 forms a recently discovered protein complex, STRIPAK, with other complex members including Protein Phosphatase-2 (PP2), STRIP, SIKE1 and certain PP2 regulatory proteins. The STRIPAK regulates multiple cell functions including gene transcription, endo- and exocytosis and cell adhesion, and several other important cell functions in cancer development and progression. The constitute members of the striatin protein complex may thus have important clinical bearings. In the present study, we investigated the expression profile of the striatin family members and key members of their interacting proteins, and discerned the relationship between the expression and disease progression and clinical outcome. Methods. Using an established fresh frozen breast cancer tissues cohort that included both normal mammary tissues and cancer tissues, we quantitatively evaluated the transcript expression of striatin-1 (STRN), striatin-3 (STRN3), straitin-4 (STRN4), its key regulator calmodulin (CALM) and the protein complex regulators protein phosphatase-2A (PPP2A), -2B (PPP2B) and the PPP2 regulatory elements PPP2R4 and PPP2R1A. The expression of each molecule was assessed against the clinical, pathological and prognostic factors of the patients. The integrated pattern of the complex members were also tested against the clinical outcome. Results. All three striatin members were expressed at good levels in mammary tissues and cancer tissues. STRN had little significant value against clinical and pathological factors. STRN3 and STRN4 were seen at high levels in tumours of high grade, with node positivity and with breast cancer related incidence. It was high level expressions of STRN3, STRN4 and CALM that were respectively associated with shorter overall survival (OS) of the patients and together they formed a poor prognostic indicator (p=0.034, HR=1.7). STRN had little impact on clinical outcomes. In a clear contrast, high levels of PPP2A, PPP2B and PPPR1A, but not PPP2R4, were seen in patients with significantly longer OS and together form a favourable prognostic indicator (p=0.034, HR=0.685). Integration of both STRN group and PPP2 group indicators constitutes a highly significant prognostic indicator for OS (p< 0.00001, HR=2.1 (95%CI 1.36-3.07)) and DFS (p=0.003, HR=1.402 (95%CI 1.12-1.75)). The predictive value of the integrated profile is independent of other clinical, pathological and hormone receptor status in multivariate analyses with OS (p< 0.0001, HR=3.861) and for DFS (p< 0.001, HR=2.055 (95%CI 1.36-3.07)). The same value stands when applied for the subtypes including triple negative breast cancers. Discussion. The STRIPAK complexes including Striatins play important regulatory roles in various cell functions and cancer development. Consistently, we found that high level expressions of STRN3, STRN4 and CALM, as a poor prognostic indicator were associated with shorter overall survival (OS) of the patients, while high levels of PPP2A, PPP2B and PPPR1A, as a favourable prognostic indicator, were seen in patients with significantly longer OS. Combination of both STRN group and PPP2 group indicators constitutes a greater significant prognostic indicator for OS and DFS. Future studies should focus on investigating the exact roles of the STRIPAK in cancer development and progression. Citation Format: Amber Xinyu Li, Andrew J. Sanders, Tracey A. Martin, Lin Ye, Fiona Ruge, QingPing Dou, Eleri Davies, Wen G. Jiang. Striatin and its interactive proteins Protein Phosphatase-2 (PP2) and PP2 regulatory elements in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-33.

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a cell–cell adhesion protein conferring heterotypic and homotypic interactions between cells of the same type and different types. It is aberrantly expressed in various cancer types and has been shown to be a regulator of cancer metastasis. In the present study, we investigated potential roles of ALCAM in the peritoneal transcoelomic metastasis in gastrointestinal cancers, a metastatic type commonly occurred in gastro-intestinal and gynaecological malignancies and resulting in poor clinical outcomes. Specifically, we studied whether ALCAM acts as both a ‘seed’ receptor in these tumour cells and a ‘soil’ receptor in peritoneal mesothelial cells during cancer metastasis. Gastric cancer and pancreatic cancer tissues with or without peritoneal metastasis were compared for their levels of ALCAM expression. The impact of ALCAM expression in these tumours was also correlated to the patients’ clinical outcomes, namely peritoneal metastasis-free survival. In addition, cancer cells of gastric and pancreatic origins were used to create cell models with decreased or increased levels of ALCAM expression by genetic knocking down or overexpression, respectively. Human peritoneal mesothelial cells were also genetically transfected to generate cell models with different profiles of ALCAM expression. These cell models were used in the tumour-mesothelial interaction assay to assess if and how the interaction was influenced by ALCAM. Both gastric and pancreatic tumour tissues from patients who developed peritoneal metastases had higher levels of ALCAM transcript than those without. Patients who had tumours with high levels of ALCAM had a much shorter peritoneal metastasis free survival compared with those who had low ALCAM expression (p = 0.006). ALCAM knockdown of the mesothelial cell line MET5A rendered the cells with reduced interaction with both gastric cancer cells and pancreatic cancer cells. Likewise, levels of ALCAM in both human gastric and pancreatic cancer cells were also a determining factor for their adhesiveness to mesothelial cells, a process that was likely to be triggered the phosphorylation of the SRC kinase. A soluble ALCAM (sALCAM) was found to be able to inhibit the adhesiveness between cancer cells and mesothelial cells, mechanistically behaving like a SRC kinase inhibitor. ALCAM is an indicator of peritoneal metastasis in both gastric and pancreatic cancer patients. It acts as not only a potential peritoneal ‘soil’ receptor of tumour seeding but also a ‘soil’ receptor in peritoneal mesothelial cells during cancer metastasis. These findings have an important therapeutic implication for treating peritoneal transcoelomic metastases.

Colorectal cancer is a serious threat to human health. Poor prognosis and frequently reported drug resistance urges research into novel biomarkers and mechanisms to aid in the understanding of the development and progression of colorectal cancer and to optimise therapeutic strategies. In the current study, we investigated the roles of a putative tumour suppressor, EPLIN, in colorectal cancer. Our clinical colorectal cancer cohort and online databases revealed a downregulation of EPLIN in colorectal cancer tissues compared with normal tissues. The reduced expression of EPLIN was associated with poor clinical outcomes of patients. In vitro cellular function assays showed that EPLIN elicited an inhibitory effect on cellular growth, adhesion, migration and invasion. Utilising a protein microarray on protein samples from normal and tumour patient tissues suggested HSP60, Her2 and other signalling events were novel potential interacting partners of EPLIN. It was further revealed that EPLIN and HSP60 were negative regulators of Her2 in colorectal cancer cells. The clinical cohort also demonstrated that expression of HSP60 and Her2 affected clinical outcomes, but most interestingly the combination of EPLIN, HSP60 and Her2 was able to identify patients with the most unfavourable clinical outcome by independently predicting patient overall survival and disease free survival. Furthermore, EPLIN and HSP60 exhibited potential to regulate cellular response to chemotherapeutic and EGFR/Her2 targeted therapeutic agents. In conclusion, EPLIN is an important prognostic factor for patients with colon cancer and reduced EPLIN in CRC contributes to aggressive traits of CRC cells and their responses to chemotherapeutic drugs. Collectively, EPLIN is a pivotal factor for the development and progression of colorectal cancer and has important clinical and therapeutic values in this cancer type.

Background/Aim: Capillary morphogenesis gene 2 (CMG2) is involved in prostate and breast cancer progression. This study aimed to investigate sex hormone receptor-mediated regulation of CMG2 in breast and prostate cancer, and its implication in disease progression. Materials and Methods: Expression of CMG2, oestrogen receptor (ER) and androgen receptor (AR) was determined in breast and prostate cancer cell lines, respectively, using real-time quantitative PCR (QPCR) and western blot. Association between CMG2 and sex hormone receptors was analysed in a number of transcriptome datasets. Immunochemical staining was performed in tissue microarrays of breast cancer (BR1505D) and prostate cancer (PR8011A). CMG2 expression was determined in 17β-oestradiol treated breast cancer cells and AR over-expressing prostate cancer cells. Results: CMG2 was found to be inversely correlated with sex hormone receptors in breast and prostate cancer. Lower expression of CMG2 was associated with a poor prognosis in ER (+) breast cancer but not ER (–) tumours. Both ER (+) breast cancer cell lines and AR (+) prostate cancer cell lines presented lower expression of CMG2, which was increased following sex hormone deprivation. Exposure to 17-β-oestradiol and AR over-expression repressed CMG2 expression in breast cancer and prostate cancer cell lines, respectively. Conclusion: CMG2 is inversely correlated with ER and AR status in breast and prostate cancer, respectively. ER and AR mediate repression of CMG2 expression in corresponding cancerous cells.

Death associated protein‑3 (DAP3) was identified as a responsive protein to interferon‑gamma‑induced cell death which possibly exerts this regulation by interacting with DAP3 binding cell Death enhancer‑1 (DELE1), a newly discovered mitochondrial stress protein in response to cell stress signals. Whilst DAP3 has been shown to be aberrantly expressed in several cancer types (i.e. breast cancer), little is known about the relationship between DAP3 and DELE1 in cancers. The present study examined the expression levels of both DAP3 and DELE1 in clinical colorectal cancers (CRCs), as well as their implication on chemoresistance and mechanism behind the action. Firstly, transcript levels of both DAP3 and DELE1 were quantitatively assessed in a clinical cohort of CRC (n=94). Tumour tissues had significantly higher levels of DAP3, but not DELE1 compared with normal tissues. Levels of DAP3 and DELE1 had a significant association with patient's clinical outcomes and local recurrence. DAP3 and DELE1 significantly correlated in normal colorectal tissues but not in tumour tissues. Secondly, the protein levels of DAP3 and DELE1 were evaluated in both normal and tumour colon tissues which showed that both proteins were highly aberrant in CRC tissues. In addition, both DAP3 and DELE1 at transcript and protein levels were identified as prognostic factors for patient's clinical outcomes. Furthermore, in

Background: Current and existing ablation for cancer treatment such as laser, RF and MW, all induce local high temperatures. The hyperthermia generated by these techniques lead to burns, pain and unwanted side effects in patients. Consequently, we aimed to develop a normothermic pulsed electric field exposure (PEFE) treatment that when applied in cancer treatments can effectively induce cell death and tumour degeneration without associated physical consequences. Method: The pulsed electric field exposure (PEFE) was generated with a carrier signal at 5.8GHz, then transmitted by a pointed open-ended coaxial cable. Breast cancer cells were treated with PEFE and assessed for their viability, apoptosis and other biological responses, together with the intracellular signalling events by using protein kinase array and related technologies. Breast cancer cells were also assessed for their ultrastructure changes by electron microscopy. In vivo experimentation was used to determine efficacy using human breast cancer tumour models in which tumours were treated with short PEFE pulses. The temperature at the area of treatment was monitored to ensure normothermic conditions. Tumour size were checked every 3 days for 36 days duration following different treatment protocols, followed by histological and biochemical analyses of the tumour and surrounding tissues. Results: MDA-MB-231 cancer cells were subject to PEFE, which induced no temperature change. Cells react to different pulse regimes and is related to cell type and aggressiveness. The treated cells became apoptotic after 4 hrs and this continued for at least 24 h after exposure with a 70% death rate. The apoptotic changes were confirmed by both immunofluorescent based and electron microscopy based methods. Protein kinase array (Kinexustm) revealed that PEFE exposure resulted in marked signature changes in the intracellular signalling event leading to apoptosis, including (BCL2, the CHK family, EGFR and ERK). In the in vivo trial, tumours were allowed to reach approximately 0.5 cm3 before treatment with PEFE with multiple conditions, which all resulted in a significant reduction of tumour volume after one week (p Citation Format: Tracey Martin, Zhe Zhao, Chang Gong, Shuo Cai, Fiona Ruge, Philippa Young, Robert E Mansel, Johannes Benedikt, Wen G Jiang. Pulsed electric field exposure (PEFE) applied in breast cancer: A potential normothermic clinical cancer therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-21.

Noggin is an antagonist of bone morphogenetic proteins (BMP), being indispensable for certain developmental events. The present study aimed to examine the role of Noggin in the development and prognosis of gastric cancer (GC) and to elucidate the underlying mechanisms. The expression of Noggin in GC was evaluated by RT‑qPCR, immunohistochemistry and by the analyses of publicly available databases. The effects of Noggin on proliferation, cell cycle, adhesion, invasion, colony formation and tumour spheroid were examined following both the knockdown and overexpression of Noggin in GC cell lines. The involvement of epidermal growth factor receptor (EGFR) signalling was examined by western blot analysis and by using small molecule inhibitors. As a result, a higher expression of Noggin in GC was found to be associated with a poorer overall survival. Noggin overexpression promoted the proliferation and colony formation of GC cells by promoting cell cycle progression. The knockdown of Noggin in HGC27 cells exerted an opposite effect on proliferation, colony formation and cell cycle progression. Noggin expression positively correlated with EGFR expression in both GC cell line models and The Cancer Genome Atlas human GC cohort. Targeting EGFR and its downstream pathways diminished cell proliferation which was promoted by Noggin. Furthermore, Noggin overexpression resulted in an enhanced nuclear translocation of β‑catenin, leading to an upregulation of EGFR. Thus, the findings of the present study demonstrate that Noggin promotes the proliferation of GC cells by upregulating EGFR and enhancing a vicious circle formed by β‑catenin, EGFR, ERK and Akt.

Introduction: Brain metastatic disease (BMD) is attributed to significant mortality and morbidity in breast cancer patients. Strict policing of molecular traffic across the blood-brain barrier (BBB) precludes brain parenchymal penetration of most small molecule drugs, chemotherapy included. Consequently, the treatment of BMD remains difficult, particularly if the BBB remains intact during disease progression. Artemisinin has been efficacious in treating multi-drug resistant cerebral malaria likely indicative of an ability to traverse the BBB. Thus, Artemisinin may be able to modulate the restrictive nature of the BBB and provide a potential adjunctive therapy to established drug therapy in the treatment of BMD. Emerging evidence suggests that Artemisinin itself may harbour potent anti-cancer properties. Aim: To investigate in vitro the effect on barrier function in particular paracellular permeability of brain vascular endothelial cells compared to non-brain vascular endothelial cells when treated with Artemisinin. Materials and Methods: Inter-individual BBB differences were modelled using the human brain vascular endothelial cell lines hCMEC-D3, TY09 and TY10. In addition to the brain cell lines, a HUVEC cell line was treated with incremental concentrations of artemisinin. Adhesion, migration and electrical resistance across cellular monolayers were quantified using Electric Cell-Substrate Impedance Sensing (ECIS). Trans- Endo/Epithelial Resistance (TEER) and Paracellular Permeability (PCP) were used to assess barrier function. Immunofluorescence was used to qualitatively assess tight junction (TJ) integrity of all cell lines. RNAseq was used to determine changes in message after treatment with Artemisinin in all 4 cell lines. Results: A significant reduction in electrical resistance across all cell lines was observed when treated with 1mM Artemisinin and may be suggestive of a cytotoxic concentration. In comparison when treated with 0.01mM Artemisinin the electrical resistance decreased which could suggest restrictive barrier properties were enhanced, in addition evidencing Artemisinin may exhibit biphasic activity. The reduction in electrical resistance across the brain endothelial cell lines was significant (p Conclusion: The results demonstrate that Artemisinin may have the capacity to transiently increase or decrease the permeability of the BBB. Therefore, Artemisinin may have a role in facilitating drug delivery to the cerebral parenchyma providing potential therapeutic benefit for the treatment of BMD or other related neurological conditions difficult to treat due to the BBB. Citation Format: Emily Appadurai, Fiona Ruge, Andrew Sanders, Wen G Jiang, Tracey A. Martin. Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-04.

Introduction: WounD14 (WD14) gene signature is a recently developed tool derived from genetic interrogation of wound edge biopsies of chronic venous leg ulcers to identify heard-to-heal wounds and enable clinicians to target aggressive therapies to promote wound healing. This study aimed to evaluate if changes in wound clinical healing status were detected by the WD14 gene signature over time as this is currently poorly understood. Material and methods: WD14 was developed through gene screening and subsequent validation in 3 patient cohorts involving 85 consecutive patients with chronic venous leg ulcers referred to a tertiary wound healing unit. Patients underwent a wound edge biopsy to interrogate for a “healing” or “non-healing” genotype. A smaller cohort (18%) underwent a second biopsy, which comprised this pilot cohort reported herein. Twelve weeks following biopsy, wounds were clinically assessed for healing status based on reduction in size and compared to WD14 genotype. Results: Sequential biopsies and WD14 scores were derived from 16 patients. WD14 signature predicted wound healing status among this cohort at either visit (32 wound edge biopsies) with a positive predictive value (PPV) of 85.2% (95% CI 74.1%-92.0%) and negative predictive value (NPV) of 80.0% (95% CI 34.2%-96.9%). A total of 6 wounds underwent altered clinical status between the 2 visits. In this cohort, WD14 has a PPV of 66.7% (95% CI 47.3%-81.7%) and NPV of 100%. Conclusion: Although the WD14 gene signature did change with wound healing status, larger studies are required to precisely clarify its role and ability to prognosticate wounds of differing clinical status over time.

Activated leukocyte cell adhesion molecule (ALCAM, or CD166) is a cell adhesion molecule and one of potential tumour metastasis ‘soil’ receptors that via homotypic and heterotypic interactions, mediates cancer cell adhesion. The present study investigated clinical, pathological and prognostic values of ALCAM in patients with pancreatic cancer. Human pancreatic cancer (PANC-1 and Mia PaCa-2) and human vascular endothelial cell lines were used to construct cell models differentially expressing levels of ALCAM. Tumour-endothelial interaction and tumour migration were assessed by a DiI-based method and electric cell-substrate impedance sensing (ECIS) assay. Pancreatic cancer tissues (n=223), collected immediately after surgery, were analysed for levels of the ALCAM transcripts, which were also analysed against clinical, pathological and clinical outcomes of the patients. ALCAM protein was assessed by immunohistochemistry on a tissue array. Our study demonstrate that pancreatic cancer tissues had significantly higher levels of ALCAM transcripts than normal tissues (P

Graphical abstract, DEAD/H box helicases are implicated in lung cancer but have not been systematically investigated for their clinical significance and function. In this study, we aimed to evaluate the potential of DEAD/H box helicases as prognostic biomarkers and therapeutic targets in lung cancer by integrated bioinformatic analysis of multivariate large-scale databases. Survival and differential expression analysis of these helicases enabled us to identify four biomarkers with the most significant alterations. These were found to be the negative prognostic factors DDX11, DDX55 and DDX56, and positive prognostic factor DDX5. Pathway enrichment analysis indicates that MYC signalling is negatively associated with expression levels of the DDX5 gene while positively associated with that of DDX11, DDX55 and DDX56. High expression levels of the DDX5 gene is associated with low mutation levels of TP53 and MUC16, the two most frequently mutated genes in lung cancer. In contrast, high expression levels of DDX11, DDX55 and DDX56 genes are associated with high levels of TP53 and MUC16 mutation. The tumour-infiltrated CD8 + T and B cells positively correlate with levels of DDX5 gene expression, while negatively correlate with that of the other three DEAD box helicases, respectively. Moreover, the DDX5-associated miRNA profile is distinguished from the miRNA profiles of DDX11, DDX55 and DDX56, although each DDX has a different miRNA signature. The identification of these four DDX helicases as biomarkers will be valuable for prognostic prediction and targeted therapeutic development in lung cancer.

Background: Chemoresistance remains one of the obstacles to overcome in the treatment of breast cancer. S100 calcium-binding protein P (S100P) has been observed to be overexpressed in several cancers and has been associated with drug resistance, metastasis, and prognosis. However, the role of S100P in chemoresistance in breast cancer has not been thoroughly determined. Methods: Immunohistochemistry was used to evaluate the expression level of S100P protein in 22 pairs (pre-chemo and post-chemo) of breast cancer tissue from patients who underwent neoadjuvant chemotherapy. The influence of S100P on the biological behavior and chemosensitivity of breast cancer cells was then investigated. Results: The protein level of S100P in breast cancer tissue was significantly higher than in benign fibroadenoma (p

Psoriasin, otherwise known as S100A7, is a member of the S100 protein family. With the key function of binding calcium, it is able to regulate a range of cellular functions. Altered Psoriasin expression is associated with poor clinical outcomes in several solid cancers. The present study aimed to examine the implication of Psoriasin in bladder cancer (BC). Expression of Psoriasin was examined in BC cell lines using PCR. Immunohistochemical (IHC) staining of Psoriasin was performed on a bladder disease spectrum tissue array. Plasmids were constructed to effectively knockdown and overexpress Psoriasin in BC cells and further utilized for in vitro BC cellular function assays. Association between Psoriasin expression and survival of patients with BC was evaluated using Kaplan-Meier survival analysis. Psoriasin was revealed to be expressed by both bladder epithelia and cancer cells as determined by IHC. Increased expression of Psoriasin was significantly correlated with a poor overall BC patient survival. Overexpression of Psoriasin in the EJ138 cell line increased cellular proliferation, adhesion and invasion, whereas knockdown exhibited the opposite effect on cellular functions in RT112 cells. Matrix metalloprotease (MMP)9 appeared to be the most affected of the three MMPs examined in these two BC cell lines. The analysis revealed a positive correlation in BC tumours between Psoriasin and MMP9. Overall, high Psoriasin expression was correlated with poor overall survival in BC patients and promoted invasiveness of BC cells via upregulation of MMPs. Psoriasin possesses certain prognostic and therapeutic potential in BC which requires further exploration.

Background. Yangzheng Xiaoji is a formulation of Chinese medicine and has been used in the treatment of solid cancer as an adjuvant to chemotherapy by reducing the side effects to the patient. There has been evidence to show that the medicine has a direct biological role in cancer cells. In the present study, we sought to investigate the potential effects of the medicine on breast cancer cells and in particular aimed to identify the key targets and molecular pathways contributing to the anti-cancer effect of the medicine. Methods. Human breast cancer cell lines (BT549, BT20, MDA MB-231, MCF-7 and ZR 75-1) with varying invasiveness and receptor status were used. The soluble extract of Yangzheng Xiaoji, namely DME25 was used in the study. The effects of DME25 on the growth, toxicity and cellular migration were assessed. Signalling kinase changes were screened using kinase antibody array based array technologies. Kinases were also validated using phosphorylation based protein blotting. Results. Of the five breast cancer cell lines tested, Yangzheng Xiaoji extract DME25 showed little cytotoxicity over a broad range of concentrations. However, DME25 were able to markedly reduce the migration of the panel of breast cancer tested, without being toxic. Triple negative cells responded in a similar fashion with other cells. It was also noted that the adhesion of these cells were also inhibited by DME25. Using a protein kinase array, it was shown that a number of kinase complexes were inhibited by the medicine, notable ones including EGFR family kinases (reduced by 35%), Janus protein kinases (JAK) (by 57%), and Ras-related C3 botulinum toxin substrate (Rac1 or CDC42 GTPase) (by 49%) and Ribosomal protein S6 kinases (RSKs) (by 52%). Given the clinical significance of RSKs in human breast cancer, we further evaluated the role of RSK and RSK inhibitors in DME mediated cell functions and have demonstrated that both in triple negative breast cancer cells and receptor positive breast cancer cell lines, DME25 was able to synergistically enhance the effect of RSK2 inhibitor, SL1010-1, on the both the cellular migration and cell growth. Conclusion. Yangzheng Xiaoji has a broad and direct effect on the migration of breast cancer, an effect unrelated to hormone receptor status and independent of cytotoxicity. The medicine appears to target kinase pathway, particularly for the RSK kinases, suggesting an important clinical implication in the treatment of breast cancer. Citation Format: Jiang WG, Ye L, Owen S, Ruge F, Martin TA, Sanders AJ, Gao G, Wei C, Wu Y, Davies E. Signalling pathways targeted by the YangZheng Xiaoji extract and the therapeutic implications in human breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-15-04.

Background. Triple negative breast cancer (TNBC) is a challenging both in the choice of therapies and clinical outcomes. In the present study, we investigated the potential prospect of a Chinese medicine formation, ShenLingLan, shown to have benefit to patients with cancer and able to influence the biological behaviour of cancer cells, on breast cancer cells in particular the differential response from TNBC and non-TNBC cells and, on the discovery that TNBC cells were particularly sensitive to the medicine, we went on to determine the signalling and mechanism of action. Methods. A panel of three TNBC (MDA MB-231, BT20 and BT549) and a panel of three non-TNBC (MCF-7, ZR 75-1 and T47D) cells were used. A soluble extract from ShenLingLan, designated as SLDM, was utilised during this study. The proliferation, cellular migration and adhesiveness were tested using conventional and biophysical methods. Signalling profiling was conducted using a protein kinase array platform (Kinexustm). Metabolic profiling was conducted using the Seahorse platform. Expression of insulin receptor (IR) and insulin-like growth factor receptor (IGFR) gene transcripts (quantitative transcript analysis) and proteins (IHC) were conducted using a fresh breast cancer cohort and tissue array, respectively. Results. SLDM had little effects on the growth of breast cancer cells. However, it had profound inhibitory effects on the migration of both TNBC and non-TNBC cells in a concentration dependent manner. Interestingly, TNBC cells were 5-20 times more sensitive than the non-TNBC cells in their migration and cell adhesion responses to SLDM. The protein array platform further revealed that, of the wide range of protein kinases, IR and IGRR1 were the most affected in that SLDM resulted in 25-50% reduction in the phosphorylation of IR and IR substrate in TNBC cells. SLDM also caused a contrasting response in IGFR1 phosphorylation in TNBC and non-TNBC cells. Metabolically, TNBC and non-TNBC cells responded to SLDM in very different fashions. For example, in TNBC cells SLDM resulted in a significant decrease in glycolytic activities, in particular that driven by insulin (30.2±12.2 pmol/min in control, 47.7±10 pmol/min with insulin and 35.4±4.2 pmol/min with insulin/SLDM, p=0.01). There is evidence that the mitochondria oxygen consumption (OCR) was also affected by SLDM in TNBC cells (p=0.01). These changes induced by SLDM were in clear contrast to non-TNBC cells which did not respond with significant reduction. Both TNBC and non-TNBC breast cancer tissues have higher IR staining than normal mammary tissues (p Conclusion. ShenLingLan has a profound inhibitory effect on the migration and cell-matrix adhesion of TNBC cells, with marked effect on the metabolics of these cells. This effect connects with reduction of the IR and IGFR activation, mainly through the reduction in glycolysis. Together with the clinical implication of IR and IGFR in breast cancer, ShenLingLan has an important role in the treatment of breast cancer with an emphasis in TNBCs. Citation Format: Jiang WG, Owen S, Ruge F, Gao Y, Wang H, Wei C, Wu Y, Davies E. The impact of the Chinese medicine ShenLingLan on triple negative breast cancer, the metabolic and signalling pathways and clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-10.

Background. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. Neratinib has been shown to have clinical activity in HER2-amplified or overexpressed breast cancers and those with HER2 mutations. However, there are indications that it may also work on other subtypes that are not strongly positive for the receptors. The present study first screened the effects of neratinib on a range of kinase targets and identified that the Wnt signalling components are key factors that allow neratinib to interact and targets. These targets were also validated in a cohort of human breast cancer. Methods. Human breast cancer cohorts (n=124) were tested for the transcript expression of HER family including EGFR, HER2, HER3 and HER4 and a number of Wnt family members and the Wnt signalling regulators, ie. GSK3, Axin-1, Axin-2 and β-catenin. The expression patterns were analysed against the clinicopathological and survival status of the patients. Neratinib was tested on a panel of breast cancer cell lines including triple negative cells for the effects on cytoxicity, cell growth, matrix adhesiveness and cellular migration. Signalling kinase pathways were screened using an antibody based kinase array. The effect of neratinib on multiple protein kinases was tested on the cell models, together with other kinase inhibitors. Results. Neratinib had an inhibitory effect on the cellular migration and cell-matrix adhesiveness of breast cancers at non-toxic concentrations, an effect more profound with MCF-7 and T47D cell lines than with BT20 and MDA MB-231 which are negative for the ER/EGFR/HER2 receptors. Of the multiple kinase inhibitors tested, neratinib was found to exert inhibition on cell function in synergy with the Wnt/β-catenin inhibitor (FK535) and GSK3 inhibitor (TWS119). The expression of the HER and Wnt family members, Wnt Inhibitory Factor-1 and Wnt regulators varied in mammary and breast cancer tissues and in their correlation with the clinicopathological factors. Of the aberrantly expressed receptors, Wnts and Wnt regulators, HER-2 and 4 were found to significantly correlate with Wnt10b (p Conclusion. Neratinib, at non-toxic concentration levels, is a profound inhibitor of the migration and matrix adhesion of breast cancer cells, cell functions linked to the aggressiveness and metastasis of breast cancer cells. Its synergistic effects with the Wnt and GSK3 inhibitor, together with the prognostic value of the HER family/Wnt, indicate that the Wnt pathway together with the HER family forms a new molecular indicator and target when considering neratinib in the treatment patients with breast cancer. Citation Format: Owen S, Ruge F, Lalani AS, Avogadri-Connors F, Bryce RP, Davies E, Jiang WG. The pan-HER inhibitor, neratinib and wingless-type MMTVs (Wnt)/Wnt regulators in human breast cancer; a biological and clinical perspective [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-07.

Aberrant expression of nephroblastoma overexpressed (NOV) has been evident\ud in certain malignancies. In the current study, we aim to investigate the role played by\ud NOV in colorectal cancer (CRC). NOV expression was determined in a cohort of 359 CRC\ud tissues and 174 normal colorectal tissues. Its impact on CRC cells was investigated\ud using in vitro NOV knockdown and overexpression models. NOV transcripts were\ud reduced in the CRC tumours compared with the paired adjacent normal colorectal\ud tissues (p < 0.01) and was associated with distant metastases. NOV knockdown\ud resulted in increased cell proliferation and invasion of RKO cells, whilst an opposite\ud effect was seen in the HT115 NOV over expressing cells. A positive association\ud between Caspase-3/-8 and NOV was seen in NOV knockdown and overexpression\ud cell lines which contributed to the survival of serum deprived CRC cells. Further\ud investigation showed that NOV regulated proliferation, survival and invasion through\ud the JNK pathway. NOV knockdown in RKO cells reduced the responsiveness to\ud 5-Fluorouracil treatment, whilst overexpression in HT115 cells exhibited a contrasting\ud effect. Taken together, NOV is reduced in CRC tumours and this is associated with\ud disease progression. NOV inhibits the proliferation and invasion of CRC cells in vitro.\ud Inhibition of proliferation is mediated by a regulation of Caspase-3/-8, via the JNK\ud pathway, which has potential for predicting and preventing chemoresistance.

Background Chronic venous leg ulcers pose a significant burden to healthcare systems, and predicting wound healing is challenging. The aim of this study was to develop a genetic test to evaluate the propensity of a chronic ulcer to heal. Methods Sequential refinement and testing of a gene expression signature was conducted using three distinct cohorts of human wound tissue. The expression of candidate genes was screened using a cohort of acute and chronic wound tissue and normal skin with quantitative transcript analysis. Genes showing significant expression differences were combined and examined, using receiver operating characteristic (ROC) curve analysis, in a controlled prospective study of patients with venous leg ulcers. A refined gene signature was evaluated using a prospective, blinded study of consecutive patients with venous ulcers. Results The initial gene signature, comprising 25 genes, could identify the outcome (healing versus non-healing) of chronic venous leg ulcers (area under the curve (AUC) 0·84, 95 per cent c.i. 0·73 to 0·94). Subsequent refinement resulted in a final 14-gene signature (WD14), which performed equally well (AUC 0·88, 0·80 to 0·97). When examined in a prospective blinded study, the WD14 signature could also identify wounds likely to demonstrate signs of healing (AUC 0·73, 0·62 to 0·84). Conclusion A gene signature can identify people with chronic venous leg ulcers that are unlikely to heal.

Surgical resection of the primary tumor may enhance the metastasis and recurrence of cancer. The reaction of patients to surgery includes changes of the immune system, the inflammatory system and the neuroendocrine system. In the perioperative period, anesthetics are used both for anesthesia and analgesia. There are several studies showing that the progression of cancer can be influenced by many kinds of anesthetics, although most of these studies are preclinical and thus have not yet influenced clinical recommendations. This review summarizes recent studies regarding the effects of anesthetics on metastasis and recurrence of cancer.

EPLIN is frequently downregulated or lost in various cancers. The purpose of this study was to evaluate the importance of EPLIN in prostate cancer progression, with particular focus on the mechanistic implications to elucidate EPLIN's tumor suppressive function in cancer. EPLIN expression was evaluated in prostate cancer cell lines and tissues. PC-3 and LNCaP EPLINα overexpression models were generated through transfection with EPLINα sequence and EPLIN knockdown was achieved using shRNA in CA-HPV-10 cells. Functional assays were performed to evaluate cellular characteristics and potential mechanisms were evaluated using a protein microarray, and validated using western blot analysis. EPLIN expression was reduced in clinical prostate cancer sections, including hyperplasia (p ≤ 0.001) and adenocarcinoma (p = 0.005), when compared to normal prostate tissue. EPLINα overexpression reduced cell growth, migration and invasion, and influenced transcript, protein and phosphoprotein expression of paxillin, FAK and Src. EPLIN knockdown increased the invasive and migratory nature of CA-HPV-10 cells and also induced changes to FAK and Src total and/or phospho expression. Functional characterization of cellular migration and invasion in addition to FAK and Src inhibition demonstrated differential effects between control and EPLINα overexpression and EPLIN knockdown cell lines. This study highlights that EPLIN expression in prostate cancer is able to influence several aspects of cancer cell characteristics, including cell growth, migration and invasion. The mechanism of the tumor suppressive action of EPLIN remains to be fully elucidated; and this study proposes a role for EPLIN's ability to regulate the aggressive characteristics of prostate cancer cells partially through regulating FAK/Src signaling.

Graves' orbitopathy (GO) is a disfiguring/distressing, inflammatory autoimmune condition. This intractable problem is caused by expansion of the orbital contents around the eye by excessive fat generation (adipogenesis) and overproduction of extracellular matrix components, especially hyaluronan (HA) from preadipocytes/fibroblasts (PFs). Current immunosuppressive/antiinflammatory treatments are largely ineffective and have unpleasant side effects, and a better therapeutic strategy through understanding GO-associated pathological features is needed.Previously we identified depot-specific HA synthase 2 regulation (HAS2; major source of HA), which facilitates orbit-specific HA accumulation during adipogenesis, and targeting phosphatidylinositol-3-kinase/mechanistic target of rapamycin-complex-1 pathways blocked both pathological features. The current study revealed low expression levels of Forkhead box O (FOXOs; critical downstream effectors of phosphatidylinositol-3-kinase) in orbital PFs through adipogenesis compared with sc levels. We aimed to dissect the role of FOXOs in GO pathogenesis to identify nonimmunosuppressive targets for GO treatment.Human orbital and sc primary PFs were treated with small interfering RNA/chemical inhibitor (AS1842856) of FOXOs or FOXO enhancer trifluoperazine hydrochloride (TFP; Food and Drug Administration approved drug), in serum-free medium for 24 hours, or TFP treatment in adipogenic medium for 15 days.Quantitative PCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA.Substantially increased or decreased HAS2/HA production was observed by inhibiting (small interfering RNA or chemical inhibitor) or enhancing (TFP) FOXO expression, respectively. TFP treatment is also sufficient to counteract thyrotropin receptor-activated HAS2/HA production and block adipogenesis in orbital PFs.FOXOs play a crucial repressor role in the regulation of HAS2/HA production and adipogenesis in orbital PFs. Our data reveal for the first time that resetting GO-associated pathological features through drug-targeted activation of FOXOs could provide a feasible nonimmunosuppressive therapeutic strategy for GO.

Hepatocyte growth factor-like protein (HGFl) and its receptor, Recepteur d'Origine Nantais (RON), have been implicated in the development of wound chronicity. HGFl and RON expression was detected in acute wound tissue, chronic wound tissue and in normal skin using quantitative polymerase chain reaction (Q-PCR). HGFl and RON expression was also assessed in chronic healing and chronic non-healing wound tissues using Q-PCR and immunohistochemical staining. Expression was similarly detected in the HaCaT immortalized human keratinocyte cell line using reverse transcription polymerase chain reaction (RT-PCR). rhHGFl was used to assess the impact of this molecule on HaCaT cell functionality using in vitro growth assays and electric cell-substrate impendence sensing (ECIS) migration assays. HGFl and RON transcript expression were significantly increased in acute wound tissue compared to chronic wound tissue and were also elevated, though non-significantly, in comparison to normal skin. Minimal expression was seen in both healing and non-healing chronic wounds. Treatment of HaCaT cells with rhHGFl had no effect on growth rates but did enhance cell migration. This effect was abolished by the addition of a phospholipase C gamma (PLCγ) small molecule inhibitor. The increased expression of HGFl and RON in acute, healing wounds and the pro-migratory effect of HGFl in an in vitro human keratinocyte model, may indicate a role for HGFl in active wound healing.

Reciprocal interactions between cancers and the surrounding microenvironment have an important role in tumour evolution. In this study, our data suggested that through thrombospondin 1 (TSP1), tumour-associated microvessel provides a dormant niche to sustain inactive status of breast invasive ductal carcinoma (IDC) cells. TSP1 levels in the tumour stroma were negatively correlated with vascular indoleamine 2,3-dioxygenase 1 (IDO1) in IDC tissues. IDO1 is an intracellular enzyme initiating the first and rate-limited step of tryptophan breakdown. Lower stromal TSP1 levels and positive tumour vascular IDO1 staining seems to associate with poor survive of patients with IDC. IDC cells induced a significantly increase in IDO1 expression in endothelial cells (ECs). IFNγ exerts a similar effect on ECs. We hypothesized a tryptophan starvation theory that since tryptophan is essential for the synthesis of TSP1, IDO1 induce a decrease in tryptophan availability and a reduction in TSP1 synthesis in ECs, leading to overcoming the dormancy state of IDC cells and exacerbating conditions such as tumour invasion and metastasis. These findings identify a non-canonical role of IFNγ/IDO1/TSP1 axis in microvascular niche-dominated dormancy of breast invasive ductal carcinoma with a solid foundation for further investigation of therapeutic and prognostic relevance.

Background: Upregulation of heat shock protein-27 (HSP27), a key member of the HSP family, has been shown to confer resistance to chemotherapy and radiotherapy in solid tumours including breast cancer. Evidence is also emerging that HSP27 is linked to the metastatic spread of breast cancer and key cellular traits including cellular migration. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. cMET, the receptor for hepatocyte growth factor and target for cancer therapies, has been shown to be trans-phosphorylated by EGFR. In the current study, we evaluated HSP27 expression within a breast cancer cohort and its implications in cellular responsiveness to cMET inhibition and neratinib. Methods: HSP27 transcript expression was analysed in our chemotherapy naïve breast cancer cohort (n=124) using quantitative PCR (qPCR) and compared to clinic-pathological information including long-term survival over a ten year follow up period. In addition, the correlation between HSP27 and HER2 expression was explored using Spearman Rank order. High-throughput cell migration analysis was performed using ECIS, on MCF-7 control and HSP27 siRNA knockdown cells in conjunction with neratinib and PHA 665752, a small molecule cMET inhibitor. Results: In our primary breast cancer cohort, there were no significant associations between HSP27 transcript expression levels and tumour grade, TNM or estrogen receptor (ER) status. Combined survival expression analysis indicated that the worst patient prognosis was associated with high levels of both HER2 and HSP27 and high HER2 and low HSP27 whereas best patient prognosis was associated with low HER2 and low HSP27 expression. Knockdown of HSP27 in MCF7 cells brought about a reduction in cellular migration compared to the control. Additionally, this reduction was enhanced by the addition of neratinib, in a concentration dependent manner, and also cMET inhibition when individually treated. Furthermore, the greatest inhibitory effects on MCF-7 migration were seen following HSP27 knockdown and combined treatment with neratinib and PHA 665752. Conclusions: Our current data suggests that HSP27 confers low sensitivity to drugs such as neratinib and PHA 665752, particularly in relation to cellular migration and hence potentially metastasis. Therefore, the targeting of HSP27, HER2 and cMET appear to act syngeristically to regulate cellular migration in vitro. Furthermore, clinically expression of HER2 and HSP27 may serve as a prognostic marker for breast cancer survival. Hence, combination therapies that target both HSP and HER2 pathways may provide new clinical opportunities for preventing breast cancer progression. Citation Format: Owen S, Sanders AJ, Ruge F, Lalani AS, Avogadri-Connors F, Bryce RP, Jiang WG. Heat shock protein 27 (HSP27) and HER2 positively correlate in breast cancer and effect cell responsiveness to neratinib and cMET inhibitor [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-05-03.

Death-associated protein 1 (DAP1) is a member of the DAP family and has been implicated in the regulation of cell growth and death including that of cancer cells. However, the roles of DAP1 in clinical cancer and in the regulation of colorectal cancer cells are largely unknown. The present study investigated the expression profile of DAP1 in human colorectal cancer and the impact of DAP1 on apoptosis and the cellular response to 5-FU. Human colorectal cancer specimens (n=94) and human colorectal cancer cell lines HRT18 and HT115 were used. DAP1 transcript and protein were evaluated using quantitative transcript analysis and immunohistochemistry. DAP1-knockdown cells were generated using anti-DAP1 transgene. The results revealed that human colorectal cancer tissues had lower levels of DAP1 when compared with the normal tissues. The reduced levels were associated with higher Dukes' stage and lymph node metastasis. Patients with low DAP1 expression had a markedly reduced survival. Loss of DAP1 in colorectal cancer cells resulted in a gain in cellular migration and loss of their sensitivity of apoptosis to chemotherapeutic agent, 5-FU. DAP1 was found to be correlated with disease progression and long-term survival of the colorectal patients. DAP1 is also a pivotal regulator of the growth and apoptosis and cellular response to chemotherapy agents.

Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

Methods of identifying chronic wounds that will heal in a timely, coordinated fashion and those that will not, together with novel therapeutic strategies, are vital for progression in the field of wound healing. Interleukin (IL)-7 has been associated with various biological and pathological processes. The present study explored the potential role of IL-7 in wound healing. IL-7 expression levels were examined in a clinical cohort of chronic wounds using reverse transcription-quantitative polymerase chain reaction and immunohistochemical staining analysis. The impact of recombinant human IL-7 (rhIL-7) on the growth and migrational rates of HaCaT keratinocyte cells was subsequently examined using in vitro growth and electric cell-substrate impedance sensing functional assays. The mRNA expression levels of IL-7 were increased in the healed chronic wound tissue samples, compared with non-healed chronic wound tissue samples, although the difference was not statistically significant. Similarly, immunohistochemical analysis revealed a greater staining intensity of IL-7 in the healed chronic wound tissue sections compared with the non-healed tissue sections. Treatment with rhIL-7 did not affect HaCaT cell growth rates, but was shown to enhance cell migration, an effect that could be further enhanced through the addition of inhibitors of neuronal Wiskott-Aldrich syndrome protein and protein kinase B. The data of the present study suggest that the expression levels of IL-7 may be increased in healing chronic wounds, and thus IL-7 may have a role in this process, potentially through its effects on the cellular migration of keratinocytes.

Phospholipase C (PLC) regulates a number of\ud cellular behaviours including cell motility, cell transformation,\ud differentiation and cell growth. PLC plays a regulatory\ud role in cancer cells partly by acting as signalling intermediates\ud for cytokines such as EGF and interleukins. The current\ud study examined the expression of the PLC isozymes in human\ud breast cancer and corresponding clinical relevance. Transcript\ud levels of human PLC-α, -β1, -δ, -ε, and -γ1 in human breast\ud cancer tissues were quantitatively determined by real-time\ud PCR. Immunochemical staining was performed for PLC-δ.\ud The clinical relevance was analysed with clinic pathological\ud information. Mammary tissues widely expressed PLC-α, -β1,\ud -δ, -ε, and -γ1. Significantly high levels of PLC -β1 and -ε\ud were seen in breast cancer tissues in comparison with normal\ud mammary gland tissues. PLC-γ1 however, showed marginally\ud low levels in tumour tissues. No significant difference was\ud seen in the expression of the PLC isozymes in tumours with\ud lymph node metastases. Moderately and poorly differentiated\ud breast tumours (grade 2 and grade 3) had significantly higher\ud levels of PLC-γ1, compared with well differentiated tumours.\ud High levels of PLC-δ were significantly correlated with a\ud shorter disease-free survival. The altered expression of other\ud isozymes had no correlation with the survival. It is concluded\ud that mammary tissues differentially expressed PLC isozymes.\ud These isozymes have certain implications in the disease development\ud and progression, with PLC-δ showing a significant\ud correlation with shorter disease-free survival.

Cytokines play important roles in the wound healing process through various signalling pathways. The JAK-STAT pathway is utilised by most cytokines for signal transduction and is regulated by a variety of molecules, including suppressor of cytokine signalling (SOCS) proteins. SOCS are associated with inflammatory diseases and have an impact on cytokines, growth factors and key cell types involved in the wound-healing process. SOCS, a negative regulator of cytokine signalling, may hold the potential to regulate cytokine-induced signalling in the chronic wound-healing process. Wound edge tissues were collected from chronic venous leg ulcer patients and classified as non-healing and healing wounds. The expression pattern of seven SOCSs members, at the transcript and protein level, were examined in these tissues using qPCR and immunohistochemistry. Significantly higher levels of SOCS3 (P=0.0284) and SOCS4 (P=0.0376) in non-healing chronic wounds compared to the healing/healed chronic wounds were observed at the transcript level. Relocalisation of SOCS3 protein in the non-healing wound environment was evident in the investigated chronic biopsies. Thus, the results show that the expression of SOCS transcript indicated that SOCS members may act as a prognostic biomarker of chronic wounds.

The ribosomal S6 protein kinase (RSK) family is an important effector of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) that could influence tumour metastasis by phosphorylating proteins in both the nuclear and cytoplasmic compartments. Aberrant expression of RSK is evident in certain malignancies but the role played by RSK in breast cancer is still not clear. This study aimed to examine the expression of RSK in human breast cancer specimens and its role to breast cancer metastasis.The expression of RSK1 to -3 were separately examined in human breast cancer tissues (normal, n=33; cancer, n=112) using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemistry. Migration and adhesion of breast cancer cells treated with the RSK inhibitor SL0101 were investigated by electric cell impedance sensing (ECIS). The effect on growth and invasion of RSK1-3 was then investigated using in vitro models.The clinical data and immunohistochemistry revealed that expression of RSK1 and RSK3 were less in tumour tissues than normal. mRNA expression of RSK2 was negatively correlated with grade, TNM staging, and survival rate. SL0101 inhibited adhesion of the MCF-7 and MDA-231 breast cancer cell lines. SL0101 suppressed MDA-231 invasion and the alternate RSK inhibitor BRD7389 inhibited the invasion of MCF-7 and MDA-231 cells.RSK1 and 3 but not RSK2 are down-regulated in breast tumour and are associated with disease progression. RSK may be a key component in the progression and metastasis of breast cancer.

Interleukin (IL)-24, also known as melanoma differentiation-associated gene-7, is a cytokine initially identified from cancerous cells and expressed in a range of cell types. It is a regulator of cell differentiation, growth, and apoptosis, and a promising anticancer agent. IL-24 acts via its heterodimic receptors: the IL-20R1 and IL-20R2 complex and the IL-22R1 and IL-20R2 complex. There is limited information on the effect of IL-24 in wound healing. Human acute and chronic wound tissues were used to analyze the transcript levels and histological staining of IL-24 and the IL-24 receptors. The biological response of human keratinocytes to recombinant human IL-24 was evaluated using electric cell-substrate impedance sensing-based methods in conjunction with inhibitors to candidate signaling pathways. IL-24 significantly slowed the migration of keratinocytes (p = 0.01), with only a marginal effect on cellular adhesion. The inhibitory effect of IL-24 on migration was completed reversed following addition of an AKT inhibitor (p = 0.004) but not an SMAD3 pathway inhibitor. Human chronic wound tissues showed raised levels of both IL-24 (p = 0.003) and its receptor (p = 0.0305) compared with acute wound tissues. We conclude that IL-24 appears to promote wound chronicity via its inhibitory effect on the migratory behavior of human keratinocytes, mediated through an AKT-dependent pathway.

Interleukin (IL)-8 is a pro-inflammatory cytokine that has a direct effect on immune cells, including polymorphonuclear cells. Keratinocytes are a rich source of IL-8. However, there is little knowledge on the role of IL-8 in clinical wound healing and the direct biological effect of IL-8 on keratinocytes. In this study, the effect of recombinant human IL-8 (rhIL-8) on migration and adhesion was tested using HaCaT keratinocytes as a cell model. The cell functions were evaluated using impedance cell sensing. The expression of IL-8 receptor (IL-8R) transcripts in human skin and wounds (acute and chronic) was assessed using real-time transcript analysis. rhIL-8 significantly increased the migration of keratinocytes (3.5±0.3 for cells treated with IL-8 vs. 2.7±0.6 for controls; p=0.029). It is interesting to note that treatment of keratinocytes with IL-8 resulted in a marked shift in the responsive frequencies. IL-8 only resulted in a marginal increase in cell adhesion, which was particularly noticeable at high frequencies. The PLC-γ inhibitor completely eradicated the action of IL-8 on the migration of HaCaT cells. Using real time PCR, it was found that chronic wounds had significantly lower levels of the B form of the IL-8R (IL-8RB) (p=0.045) and marginally lower levels of the A form, IL-8RA, in comparison with acute wounds. Therefore, IL-8 has a direct and profound stimulatory effect on the migration of human keratinocytes, which is likely to occur via the PLC-γ pathway. Together with a reduced level of IL-8Rs in difficult-healing wounds, IL-8 has a clear prognostic and therapeutic value in wound healing.

Defolliculated (Gsdma3(Dfl)/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3(Dfl)/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1-/- mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies.

Background. The EGF receptor (EGFR) kinase family plays important role in tumour growth and progression and has been a validated therapeutic target for lung cancers. Here, we examined the activity of an irreversible pan-HER tyrosine kinase inhibitor, neratinib, on kinase phosphorylation and cellular response in human gastric cancer. The study also examined the expression of the neratinib responsive kinases in relation to chemoresistance in human gastric cancer. Method. Tumour and normal gastric tissues from a cohort of 85 patients were obtained along with clinical outcome and therapeutic response to chemotherapy. The level of expression of a panel of protein kinases of interest, including EGFR family members, was quantitatively analysed using a protein based kinase array. Human gastric cancer cells (AGS and HGC27) were assessed for their sensitivity to neratinib, inhibitors of other candidate kinases and combinations thereof. Cellular growth, migration and matrix adhesiveness were evaluated using multiple in vitro platforms. Result. We identified a panel of kinases that were highly responsive to the treatment by neratinib in cell lines. In the gastric cancer cohort, expression of these kinases significantly correlated with the overall survival of the patients (p Citation Format: W. G. Jiang, Tracey A. Martin, Sioned Owen, Lin Ye, Andrew J. Sanders, Yuxin Cui, Meng Xie, Shiqin Jia, Yongning Jia, Fiona Ruge, Francesca Avogadri-Connors, Alshad S. Lalani, Richard P. Bryce, Jiafu Ji. Investigating the activity of neratinib in human gastric cancer and gastric cancer cells, implications on clinical outcome and chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 879.