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To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

// Danielle R. Bond 1, 4 , Crystal Naudin 1, 3 , Adam P. Carroll 1, 5 , Belinda J. Goldie 1, 2 , Joshua S. Brzozowski 1 , Helen M. Jankowski 1 , Murray J. Cairns 1, 5 , Leonie K. Ashman 1 , Christopher J. Scarlett 4 and Judith Weidenhofer 1 1 School of Biomedical Science and Pharmacy, The University of Newcastle and Hunter Medical Research Institute (HMRI), NSW, Newcastle, Australia 2 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia 3 Department of Pediatrics, Emory University, Atlanta, GA, USA 4 School of Environmental and Life Sciences, The University of Newcastle and Hunter Medical Research Institute (HMRI), NSW, Newcastle, Australia 5 Schizophrenia Research Institute, Sydney, NSW, Australia Correspondence to: Judith Weidenhofer, email: judith.weidenhofer@newcastle.edu.au Keywords: miR-518f-5p; prostate cancer; CD9; transcript regulation; metastasis Received: May 26, 2017 Accepted: November 15, 2017 Published: December 07, 2017 ABSTRACT Tetraspanin CD9 is generally considered to be a metastasis suppressor, with decreased levels associated with progression and metastasis in many advanced stage cancers. Little is known about the cause of CD9 dysregulation in prostate cancer, however there are several miRNA-binding sites in the 3´UTR of the transcript suggesting it could be post-transcriptionally regulated. Using microarrays and luciferase assays in tumourigenic and non-tumourigenic prostate cell lines we identified miR-518f-5p as a regulator of the CD9 3'UTR gene expression, and decreased expression of endogenous CD9 in non-tumorigenic prostate RWPE1 and prostate cancer DU145 cells. This resulted in differential functional effects, in which RWPE1 cells showed increased migration and decreased adhesion to extracellular matrix substrates, whereas DU145 cells showed decreased migration and increased adhesion. Moreover, overexpression of miR-518f-5p significantly increased proliferation between 48h and 72h in normal RWPE1 cells, with no effect on tumourigenic DU145 cell proliferation. These results show that tetraspanin CD9 is regulated by miRNAs in prostate cell lines and that due to differential functional effects in non-tumourigenic versus tumourigenic prostate cells, miR-518f-5p may be an effective biomarker and/or therapeutic target for prostate cancer progression.

Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3&prime, UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.

Pancreatic cancer (PC) is a complex, heterogeneous disease with a dismal prognosis. Current therapies have failed to improve survival outcomes, urging the need for discovery of novel targeted treatments. Bispidinone derivatives have yet to be investigated as cytotoxic agents against PC cells. The cytotoxic effect of four bispidinone derivatives (BisP1: 1,5-diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one, BisP2: 3,7-bis-(2-(S)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride, BisP3: [2-{7-[2-(S)-tert-butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tertbutyl ester, BisP4: 3,7-bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride) was assessed against PC cell lines (MiaPaca-2, CFPAC-1 and BxPC-3). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) colorimetric assay, while apoptotic cell death was confirmed using fluorescence microscopy and flow cytometry. Initial viability screening revealed significant cytotoxic activity from BisP4 treatment (1 &micro, M&ndash, 100 &micro, M) on all three cell lines, with IC50 values for MiaPaca-2, BxPC-3, and CFPAC-1 16.9 &micro, M, 23.7 &micro, M, and 36.3 &micro, M, respectively. Cytotoxic treatment time-response (4 h, 24 h, and 48 h) revealed a 24 h treatment time was sufficient to produce a cytotoxic effect on all cell lines. Light microscopy evaluation (DAPI staining) of BisP4 treated MiaPaca-2 PC cells revealed dose-dependent characteristic apoptotic morphological changes. In addition, flow cytometry confirmed BisP4 induced apoptotic cell death induction of activated caspase-3/-7. The bispidinone derivative BisP4 induced an apoptosis-mediated cytotoxic effect on MiaPaca-2 cell lines and significant cytotoxicity on CFPAC-1 and BxPC-3 cell lines. Further investigations into the precise cellular mechanisms of action of this class of compounds are necessary for potential development into pre-clinical trials.

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis.

Current chemotherapy drugs for pancreatic cancer only offer an increase in survival of up to six months. Additionally, they are highly toxic to normal tissues, drastically affecting the quality of life of patients. Therefore, the search for novel agents, which induce apoptosis in cancer cells while displaying limited toxicity towards normal cells, is paramount. The olive biophenols, oleuropein, hydroxytyrosol and tyrosol, have displayed cytotoxicity towards cancer cells without affecting non-tumorigenic cells in cancers of the breast and prostate. However, their activity in pancreatic cancer has not been investigated. Therefore, the aim of this study was to determine the anti-pancreatic cancer potential of oleuropein, hydroxytyrosol and tyrosol. Pancreatic cancer cells (MIA PaCa-2, BxPC-3, and CFPAC-1) and non-tumorigenic pancreas cells (HPDE) were treated with oleuropein, hydroxytyrosol and tyrosol to determine their effect on cell viability. Oleuropein displayed selective toxicity towards MIA PaCa-2 cells and hydroxytyrosol towards MIA PaCa-2 and HPDE cells. Subsequent analysis of Bcl-2 family proteins and caspase 3/7 activation determined that oleuropein and hydroxytyrosol induced apoptosis in MIA PaCa-2 cells, while oleuropein displayed a protective effect on HPDE cells. Gene expression analysis revealed putative mechanisms of action, which suggested that c-Jun and c-Fos are involved in oleuropein and hydroxytyrosol induced apoptosis of MIA PaCa-2 cells.

Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 5%. Resistance to conventional treatment options and toxicity of current chemotherapy agents (gemcitabine) makes pancreatic cancer a target for the development of novel therapeutic agents. Oleuropein is the most abundant biophenol found in olive products; it has anti-atherogenic and anti-inflammatory properties as well as activity against cancers of the breast, colon and prostate. However, there has yet to be any investigation into the effects of oleuropein on pancreatic cancer cells. Consequently, this study aimed to assess the anti-pancreatic cancer activity of oleuropein in vitro. Two cell lines were investigated; a pancreatic cancer cell line (MiaPaCa-2) and a normal pancreas cell line (HPDE). The viability of cells after treatment with 0-200μM oleuropein was assessed using the Dojindo CCK-8 viability assay and compared to gemcitabine. The induction of apoptosis was measured by way of caspase 3/7 activation, using a MUSE flow cell analyser, and expression of Bcl-2, Bax and Bac using Western blot. Cell cycle analysis was conducted using the MUSE flow cell analyser. RNA expression was assessed using Affymetrix GeneChip® Whole Transcript (WT) expression arrays. The IC50 values for oleuropein against MiaPaCa-2 cells was148μM. However, importantly, oleuropein did not decrease the viability of HPDE cells within the treatment range. In comparision, 20nM of gemcitabine did not show selectivity; it reduced the viability of MiaPaCa-2 cells to 21% and of HPDE cells to 2%. An increase in the expression of caspase 3/7 was seen in MiaPaCa-2 cells when treated with oleuropein but it had no effect on the HPDE cells. Furthermore, when treated with oleuropein, an increase in the expression of genes involved in the NRF-2 (oxidative stress) pathway was observed in MiaPaCa-2 cells, an effect not observed in HPDE cells. Conclusion Oleuropein selectively induced apoptosis in the pancreatic cancer cells (MiaPaCa-2), appearing non-toxic to normal pancreas cells (HPDE) within the treatment ranges; this is significant, since gemcitabine was comparatively more toxic to HPDE cells. Furthermore, the link between oleuropein and the NRF-2 pathway in MiaPaCa-2 cells justifies further study into the mechanisms of action of oleuropein and its potential as a novel therapeutic approach for pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Chloe D. Goldsmith, Helen Jankowski, Danielle Bond, Judith Weidenhofer, Costas Stathopoulos, Paul Roach, Christopher Scarlett. The olive biophenol oleuropein selectively induces apoptosis in pancreatic cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2177. doi:10.1158/1538-7445.AM2017-2177