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BackgroundGlioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKCι. We have previously shown that PKCι is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKCι in glioblastoma.ResultsGlioblastoma cells in which PKCι was either depleted by shRNA or inhibited pharmacologically were unable to coordinate the formation of a single leading edge lamellipod. Instead, some cells generated multiple small, short-lived protrusions while others generated a diffuse leading edge that formed around the entire circumference of the cell. Confocal microscopy showed that this behavior was associated with altered behavior of the cytoskeletal protein Lgl, which is known to be inactivated by PKCι phosphorylation. Lgl in control cells localized to the lamellipod leading edge and did not associate with its binding partner non-muscle myosin II, consistent with it being in an inactive state. In PKCι-depleted cells, Lgl was concentrated at multiple sites at the periphery of the cell and remained in association with non-muscle myosin II. Videomicroscopy also identified a novel role for PKCι in the cell cycle. Cells in which PKCι was either depleted by shRNA or inhibited pharmacologically entered mitosis normally, but showed marked delays in completing mitosis.ConclusionsPKCι promotes glioblastoma motility by coordinating the formation of a single leading edge lamellipod and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKCι is required for the transition of glioblastoma cells through mitosis. PKCι therefore has a role in both glioblastoma invasion and proliferation, two key aspects in the malignant nature of this disease.

Oncolytic virus (OV) therapy has emerged as a novel tool in our therapeutic arsenals for fighting cancer. As a live biologic agent, OV has the ability to target and selectively amplify at the tumor sites. We have reported that a vaccinia-based OV (Pexa-Vec) has shown good efficacy in preclinical models and in clinical trials. To give an additional tool to clinicians to allow both treatment of the tumor and improved visualization of tumor margins, we developed new viral-based platforms with 2 specific gene reporters. Methods: We incorporated the human sodium iodide symporter (hNIS) and the human somatostatin receptor 2 (hSSR2) in the vaccinia-based OV and tested viral constructs for their abilities to track and treat tumor development in vivo. Results: Early and high-level expression of hNIS is detrimental to the recombinant virus, leading to the aggregation of hNIS protein and early cell death. Putting hNIS under a late synthetic promoter allowed a higher functional expression of the protein and much stronger 123 I or 99 Tc uptake. In vivo, the hNIS-containing virus infected and amplified in the tumor site, showing a better efficacy than the parental virus. The hNIS expression at the tumor site allowed for the imaging of viral infection and tumor regression. Similarly, hSSR2-containing OV vaccinia infected and lysed cancer cells. Conclusion: When tumor-bearing mice were given hNIS-and hSSR2-containing OV, 99 Tc and 111 In signals coalesced at the tumor, highlighting the power of using these viruses for tumor diagnosis and treatment.

Senescence is an irreversible growth arrest phenotype adopted by cells that has a key role in protecting organisms from cancer. There is now considerable interest in therapeutic strategies that reactivate this process to control the growth of cancer cells. Protein kinase C iota (PKCι) is a member of the atypical protein kinase C family and an important downstream mediator in the phosphoinositide pathway. PKCι expression was found to be upregulated in a subset of breast cancers and breast cancer cell lines. Introduction of mutant, oncogenic PIK3CA, but not wild-type PIK3CA, into breast mammary epithelial cells increased both the expression and activation of PKCι. In breast cancer cell lines overexpressing PKCι, depletion of PKCι increased the number of senescent cells, as assessed by senescence-associated β-galactosidase, morphology and bromodeoxyuridine incorporation. This phenomenon was not restricted to breast cancer cells, as it was also seen in glioblastoma cells. Senescence induction did not require p53, p16 or Arf and was not associated with activation of the DNA damage response. Depletion of PKCι had no effect on senescence in normal mammary epithelial cell lines. We conclude that PKCι is overexpressed in a subset of cancers where it functions to suppress premature senescence. This function appears to be restricted to cancer cells and inhibition of PKCι may therefore be an effective way to selectively activate premature senescence in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1224. doi:10.1158/1538-7445.AM2011-1224

identified in mouse and human squamous cell carcinoma. Analysis of macrophages phenotypes in tumour lesions shows that wild-type mice exhibited higher frequency of M2 macrophages. Conclusion Our findings indicate that the development of SCC was associated with specific and significant frequency of M2 macrophages in tumour samples. Introduction Systemic relapse is the major cause of mortality in extremity soft tissue sarcoma (ESTS). Traditional systemic therapies for metastatic sarcoma have limited efficacy, as do novel agents such as immune checkpoint inhibitors (ICI). Oncolytic virotherapy is capable of improving the efficacy of ICI in some non-sarcomatous pathology. In a rat model of ESTS, we have previously found isolated limb perfusion (ILP) to be ideally suited to delivering a vaccinia virus (GLV-1h68), alongside melphalan and tumour necrosis factor-a (TNFa). Using this model, we sought to investigate the effects of GLV1h68 delivered by ILP on the tumour microenvironment and determine whether viral ILP can sensitise sarcomas to subsequent immune checkpoint blockade. Material and methods In vivo experiments were performed in Brown Norway rats bearing BN175 sarcoma in accordance with a Home Office animal license. Therapeutics included GLV-1h68, a PD-1 inhibitor (J43), melphalan and TNFa. Results and discussions PD-1 inhibition had limited in vivo monotherapy efficacy in BN175 sarcoma. Pre-treatment with GLV-1h68 delivered by ILP prior to PD-1 blockade markedly improved therapy, with complete tumour regression in a third of animals. Without further treatment, resistant disease rapidly evolved leading to both local and distant relapse. However, when performed as a neoadjuvant treatment prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented local and distant relapse in all animals. Treatment with both GLV1h68 and PD-1 blockade were found to be necessary for durable cure. In vitro, GLV-1h68 induced ATP, calreticulin and HMGB1, markers of immunogenic cell death. In vivo, viral ILP and PD-1 blockade significantly increased the number of intra-tumoural CD4 + and CD8 + effector cells, with an increased proportion of these cells expressing activation markers. Viral ILP and PD-1 blockade also altered the topography of intra-tumoural immune invasion; significantly increasing CD8 + cells within the tumour parenchyma relative to the invasive margin. The accumulation of effector cells within regional lymph nodes, located outside of the perfusion field, was also noted indicative of at least a locoregional, and perhaps, systemic immune-priming. Conclusion Viral ILP and PD-1 blockade combine to prevent local and distant relapse in an animal model of high-grade ESTS. These data provide a strong rationale for clinical translation of this neoadjuvant combination immunotherapy. PO-360 PO-361 MODULATION OF CANCER CELL RESPONSE TO ETOPOSIDE BY M1 AND M2 POLARISED MACROPHAGESF CLEMENT*, T Arnould, C Michiels. Université de Namur, Unité de Recherche en Biologie Cellulaire, Namur, Belgium 10.1136/esmoopen-2018-EACR25...

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One in nine (11%) women is expected to develop breast cancer during their lifetime. Abnormal activation of the PI3K signaling cascade is common in breast cancer. The p110α catalytic subunit of PI3K is mutated in 20-40% of breast cancers. The two most common mutations are E545K in the helical domain and H1047R in the kinase domain; these mutations render PI3K constitutively active. PKC iota is a member of atypical class of PKC family serine/threonine kinases and a downstream effector in the PI3K signaling pathway. PKC iota has been implicated in carcinogenesis and oncogenic signaling in lung, colon and ovarian carcinomas but its role in breast cancer progression is unknown. Using immunohistochemistry (IHC), we have evaluated PKCι expression and localization in breast cancer tissue microarrays (TMA). Weak PKCι staining was detected in normal breast tissue. PKCι was over-expressed in subset of breast cancers with no staining in the surrounding stroma. Positive tumor staining was mainly cytoplasmic with nuclear staining in some cases. There was no significant correlation of positive PKCι staining with tumour type. PKCι overexpression was also seen in a subset of ductal carcinoma in situ samples. In vitro, we have also shown that PKCι is over-expressed and has higher levels of phosphorylation in a subset of breast cancer cell lines when compared to mammary epithelial cell lines. Stable cell lines expressing E545K or H1047R mutations were generated in the mammary myoepithelial cell line MCF-10A by retroviral transduction. Using Western blotting, we have shown that these mutations are sufficient to increase PKCι expression and activation. These results suggest a possible mechanism for the PKCι overexpression previously demonstrated by IHC. Two structurally independent PI3K inhibitors, Wortmannin and LY294-002, inhibited PKCι phosphorylation and activation in mutant PI3K expressing cells, confirming that the PKCι activation is PI3K-dependent. These inhibitors did not inhibit the low level of PKCι activation seen in MCF10A cells expressing wild-type PI3K control, suggesting that this activation occurs by a PI3K-independent mechanism. These results demonstrate that PKCι is overexpressed in some breast cancers, and that PI3KCA mutations may drive this overexpression. These results also indicate a potential role for PKCι in breast carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3171.