Surgery (RMH) - Research Publications

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    Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy
    Poh, AR ; Love, CG ; Chisanga, D ; Steer, JH ; Baloyan, D ; Chopin, M ; Nutt, S ; Rautela, J ; Huntington, ND ; Etemadi, N ; O'Brien, M ; O'Keefe, R ; Ellies, LG ; Macri, C ; Mintern, JD ; Whitehead, L ; Gangadhara, G ; Boon, L ; Chand, AL ; Lowell, CA ; Shi, W ; Pixley, FJ ; Ernst, M (AMER ASSOC ADVANCEMENT SCIENCE, 2022-06-24)
    Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.
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    Paracrine IL-6 Signaling Confers Proliferation between Heterogeneous Inflammatory Breast Cancer Sub-Clones
    Morrow, RJ ; Allam, AH ; Yeo, B ; Deb, S ; Murone, C ; Lim, E ; Johnstone, CN ; Ernst, M (MDPI, 2022-05-01)
    Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.
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    Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells
    Carli, ALE ; Afshar-Sterle, S ; Rai, A ; Fang, H ; O'Keefe, R ; Tse, J ; Ferguson, FM ; Gray, NS ; Ernst, M ; Greening, DW ; Buchert, M (WILEY, 2021-05-28)
    Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1OE -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1PAR ). Quantitative proteome analysis of MKN1OE -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1PAR -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.
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    Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression
    Luk, IY ; Jenkins, LJ ; Schoffer, KL ; Ng, I ; Tse, JWT ; Mouradov, D ; Kaczmarczyk, S ; Nightingale, R ; Burrows, AD ; Anderson, RL ; Arango, D ; Dopeso, H ; Croft, L ; Richardson, MF ; Sieber, OM ; Liao, Y ; Mooi, JK ; Vukelic, N ; Reehorst, CM ; Afshar-Sterle, S ; Whitehall, VLJ ; Fennell, L ; Abud, HE ; Tebbutt, NC ; Phillips, WA ; Williams, DS ; Shi, W ; Mielke, LA ; Ernst, M ; Dhillon, AS ; Clemons, NJ ; Mariadason, JM (SPRINGERNATURE, 2022-05-23)
    Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.
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    Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin
    Berthelet, J ; Foroutan, M ; Bhuva, DD ; Whitfield, HJ ; El-Saafin, F ; Cursons, J ; Serrano, A ; Merdas, M ; Lim, E ; Charafe-Jauffret, E ; Ginestier, C ; Ernst, M ; Hollande, F ; Anderson, RL ; Pal, B ; Yeo, B ; Davis, MJ ; Merino, D (MDPI, 2022-05-01)
    The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine.
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    STAT3 Signaling in Breast Cancer: Multicellular Actions and Therapeutic Potential.
    To, SQ ; Dmello, RS ; Richards, AK ; Ernst, M ; Chand, AL (MDPI AG, 2022-01-15)
    Interleukin (IL)-6 family cytokines, such as IL-6 and IL-11, are defined by the shared use of the gp130 receptor for the downstream activation of STAT3 signaling and the activation of genes which contribute to the "hallmarks of cancer", including proliferation, survival, invasion and metastasis. Increased expression of these cytokines, or the ligand-specific receptors IL-6R and IL-11RA, in breast tumors positively correlate to disease progression and poorer patient outcome. In this review, we examine evidence from pre-clinical studies that correlate enhanced IL-6 and IL-11 mediated gp130/STAT3 signaling to the progression of breast cancer. Key processes by which the IL-6 family cytokines contribute to the heterogeneous nature of breast cancer, immune evasion and metastatic potential, are discussed. We examine the latest research into the therapeutic targeting of IL-6 family cytokines that inhibit STAT3 transcriptional activity as a potential breast cancer treatment, including current clinical trials. The importance of the IL-6 family of cytokines in cellular processes that promote the development and progression of breast cancer warrants further understanding of the molecular basis for its actions to help guide the development of future therapeutic targets.
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    Onco-miR-21 Promotes Stat3-Dependent Gastric Cancer Progression.
    Tse, J ; Pierce, T ; Carli, ALE ; Alorro, MG ; Thiem, S ; Marcusson, EG ; Ernst, M ; Buchert, M (MDPI AG, 2022-01-06)
    MicroRNA-21 (miR-21) is a small, non-coding RNA overexpressed in gastric cancer and many other solid malignancies, where it exhibits both pro-and anti-tumourigenic properties. However, the pathways regulating miR-21 and the consequences of its inhibition in gastric cancer remain incompletely understood. By exploiting the spontaneous Stat3-dependent formation of inflammation-associated gastric tumors in Gp130F/F mice, we functionally established miR-21 as a Stat3-controlled driver of tumor growth and progression. We reconciled our discoveries by identifying several conserved Stat3 binding motifs upstream of the miR-21 gene promoter, and showed that the systemic administration of a miR-21-specific antisense oligonucleotide antagomir reduced the established gastric tumor burden in Gp130F/F mice. We molecularly delineated the therapeutic benefits of miR-21 inhibition with the functional restoration of PTEN in vitro and in vivo, alongside an attenuated epithelial-to-mesenchymal transition and the extracellular matrix remodeling phenotype of tumors. We corroborated our preclinical findings by correlating high STAT3 and miR-21 expression with the reduced survival probability of gastric cancer patients. Collectively, our results provide a molecular framework by which miR-21 mediates inflammation-associated gastric cancer progression, and establish miR-21 as a robust therapeutic target for solid malignancies characterized by excessive Stat3 activity.
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    Exploiting the STAT3 Nexus in Cancer-Associated Fibroblasts to Improve Cancer Therapy.
    Allam, A ; Yakou, M ; Pang, L ; Ernst, M ; Huynh, J (Frontiers Media SA, 2021)
    The tumor microenvironment (TME) is composed of a heterogenous population of cells that exist alongside the extracellular matrix and soluble components. These components can shape an environment that is conducive to tumor growth and metastatic spread. It is well-established that stromal cancer-associated fibroblasts (CAFs) in the TME play a pivotal role in creating and maintaining a growth-permissive environment for tumor cells. A growing body of work has uncovered that tumor cells recruit and educate CAFs to remodel the TME, however, the mechanisms by which this occurs remain incompletely understood. Recent studies suggest that the signal transducer and activator of transcription 3 (STAT3) is a key transcription factor that regulates the function of CAFs, and their crosstalk with tumor and immune cells within the TME. CAF-intrinsic STAT3 activity within the TME correlates with tumor progression, immune suppression and eventually the establishment of metastases. In this review, we will focus on the roles of STAT3 in regulating CAF function and their crosstalk with other cells constituting the TME and discuss the utility of targeting STAT3 within the TME for therapeutic benefit.
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    Multicellular Effects of STAT3 in Non-small Cell Lung Cancer: Mechanistic Insights and Therapeutic Opportunities.
    Parakh, S ; Ernst, M ; Poh, AR (MDPI AG, 2021-12-11)
    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of lung cancer cases. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) is frequently observed in NSCLC and is associated with a poor prognosis. Pre-clinical studies have revealed an unequivocal role for tumor cell-intrinsic and extrinsic STAT3 signaling in NSCLC by promoting angiogenesis, cell survival, cancer cell stemness, drug resistance, and evasion of anti-tumor immunity. Several STAT3-targeting strategies have also been investigated in pre-clinical models, and include preventing upstream receptor/ligand interactions, promoting the degradation of STAT3 mRNA, and interfering with STAT3 DNA binding. In this review, we discuss the molecular and immunological mechanisms by which persistent STAT3 activation promotes NSCLC development, and the utility of STAT3 as a prognostic and predictive biomarker in NSCLC. We also provide a comprehensive update of STAT3-targeting therapies that are currently undergoing clinical evaluation, and discuss the challenges associated with these treatment modalities in human patients.
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    Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer.
    Pang, L ; Ernst, M ; Huynh, J (MDPI AG, 2021-10-12)
    Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8α, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues.