Krista Vincent

COX‐2 Induces Breast Cancer Stem Cells via EP4/PI3K/AKT/NOTCH/WNT Axis

Cancer stem‐like cells (SLC) resist conventional therapies, necessitating searches for SLC‐specific targets. We established that cyclo‐oxygenase(COX)‐2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E‐2 receptor EP4. Present study revealed that COX‐2 induces SLCs by EP4‐mediated NOTCH/WNT signaling. Ectopic COX‐2 over‐expression in MCF‐7 and SKBR‐3 cell lines resulted in: increased migration/invasion/proliferation, epithelial‐mesenchymal transition (EMT), elevated SLCs (spheroid formation), increased ALDH activity and colocalization of COX‐2 and SLC markers (ALDH1A, CD44, β‐Catenin, NANOG, OCT3/4, SOX‐2) in spheroids. These changes were reversed with COX‐2‐inhibitor or EP4‐antagonist (EP4A), indicating dependence on COX‐2/EP4 activities. COX‐2 over‐expression or EP4‐agonist treatments of COX‐2‐low cells caused up‐regulation of NOTCH/WNT genes, blocked with PI3K/AKT inhibitors. NOTCH/WNT inhibitors also blocked COX‐2/EP4 induced SLC induction. Microarray analysis showed up‐regulation of numerous SLC‐regulatory and EMT‐associated genes. MCF‐7‐COX‐2 cells showed increased mammary tumorigenicity and spontaneous multiorgan metastases in NOD/SCID/IL‐2Rγ‐null mice for successive generations with limiting cell inocula. These tumors showed up‐regulation of VEGF‐A/C/D, Vimentin and phospho‐AKT, down‐regulation of E‐Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF‐7‐COX‐2 cells also showed increased lung colonization in NOD/SCID/GUSB‐null mice, an effect reversed with EP4‐knockdown or EP4A treatment of the MCF‐7‐COX‐2 cells. COX‐2/EP4/ALDH1A mRNA expression in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. In situ immunostaining of human breast tumor tissues revealed co‐localization of SLC markers with COX‐2, supporting COX‐2 inducing SLCs. High COX‐2/EP4 mRNA expression was linked with reduced survival. Thus, EP4 represents a novel SLC‐ablative target in human breast cancer. Stem Cells 2016;34:2290–2305

Mechanism of inhibition of calcium oxalate crystal growth by an osteopontin phosphopeptide

Osteopontin (OPN) inhibits the nucleation and/or growth of several biominerals, including hydroxyapatite (HA) and calcium oxalate monohydrate (COM), and is thought to function in the prevention of soft-tissue calcification. In previous studies, pOPAR, a peptide corresponding to amino acids 65–80 of rat bone OPN (pSHDHMDDDDDDDDDGD), was shown to be a potent inhibitor of HA crystal growth. We now show that formation of COM in the presence of this peptide results in plate-shaped crystals with rounded ends and scalloped {100} faces. Measurement of crystal dimensions revealed that the pOPAR inhibits growth of COM faces in the order {100} > {121} > {010}. Crystal growth inhibitors are believed to act by adsorbing to growth steps, sites at which lattice-ion addition is energetically favoured. To test this hypothesis, we performed molecular dynamics (MD) simulations of pOPAR adsorption to {121} steps on a {100} face and {121} steps on an {010} face. In the former case, the peptide adsorbs to the {100} (terrace) plane in preference to the {121} (riser) plane; in the latter, the peptide adsorbs to the {121} (riser) plane in preference to the {010} (terrace) plane. These studies represent the first use of MD to study the interaction between an inhibitor and crystal steps. Contrary to the prevailing belief that crystal growth inhibitors adsorb to both lattice planes of a step, we show that pOPAR interacts preferentially to either the terrace or the riser, depending on which is more cationic.

A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

On the catalysis of calcium oxalate dihydrate formation by osteopontin peptides.

Inhibition of calcium oxalate monohydrate (COM) formation and initiation of the dihydrate (COD) phase by osteopontin (OPN) have been proposed to play an important role in preventing kidney stone formation. We have studied the roles of OPN phosphate and carboxylate groups in the modulation of calcium oxalate (CaOx) crystallization using synthetic peptides corresponding to residues 65-80, 129-144, 220-235 and 273-288 of rat OPN. We investigated the effects of these peptides (0-20 μg/ml) on COM and COD formation by correlating qualitative and quantitative microscopic data with the physicochemical characteristics of the peptides used. In general, highly acidic/hydrophilic peptides strongly inhibit COM and promote COD formation. However, OPN129-144, which is basic, and OPN273-288, which is only slightly acidic, also induced COD precipitation. It is likely that inhibition of nucleation and/or growth of COM by OPN peptides maintains a high supersaturation, thereby allowing formation of the more-soluble dihydrate polymorph. In addition, growth of COD from the substrate in <100>/<110> directions suggests that highly acidic OPN peptides may nucleate crystals from the Ca(2+)-rich {100}/{110} faces. At higher peptide concentrations, however, peptides containing either phosphates or contiguous carboxylates inhibit COD, whereas peptides containing both promote COD formation further.

Assessing breast cancer cell lines as tumour models by comparison of mRNA expression profiles

IntroductionBreast cancer researchers use cell lines to model myriad phenomena ranging from DNA repair to cancer stem cell phenotypes. Though appropriate, and even requisite, for many studies, the suitability of cell lines as tumour models has come into question owing to possibilities of tissue culture artefacts and clonal selection. These issues are compounded by the inability of cancer cells grown in isolation to fully model the in situ tumour environment, which also contains a plethora of non-tumour cell types. It is thus important to understand similarities and differences between cancer cell lines and the tumours that they represent so that the optimal tumour models can be chosen to answer specific research questions.MethodsIn the present study, we compared the RNA-sequencing transcriptomes of a collection of breast cancer cell lines to transcriptomes obtained from hundreds of tumours using The Cancer Genome Atlas. Tumour purity was accounted for by analysis of stromal and immune scores using the ESTIMATE algorithm so that differences likely resulting from non-tumour cells could be accounted for.ResultsWe found the transcriptional characteristics of breast cancer cell lines to mirror those of the tumours. We identified basal and luminal cell lines that are most transcriptionally similar to their respective breast tumours. Our comparison of expression profiles revealed pronounced differences between breast cancer cell lines and tumours, which could largely be attributed to the absence of stromal and immune components in cell culture. A focus on the Wnt pathway revealed the transcriptional downregulation or absence of several secreted Wnt antagonists in culture. Gene set enrichment analysis suggests that cancer cell lines have enhanced proliferation and glycolysis independent of stromal and immune contributions compared with breast cancer cells in situ.ConclusionsThis study demonstrates that many of the differences between breast cancer cell lines and tumours are due to the absence of stromal and immune components in vitro. Hence, extra precautions should be taken when modelling extracellular proteins in vitro. The specific differences discovered emphasize the importance of choosing an appropriate model for each research question.

CCN2 Expression by Tumor Stroma Is Required for Melanoma Metastasis

Metastatic melanoma has an extremely poor prognosis with few durable remissions. The secreted matricellular protein connective tissue growth factor (CCN2) is overexpressed in cancers including melanoma and may represent a viable therapeutic target. However, the mechanism underlying the contribution of CCN2 to melanoma progression is unclear. Herein, we use the highly metastatic murine melanoma cell line B16(F10) and syngeneic mice, in which CCN2 expression is knocked out in fibroblasts, to demonstrate that loss of CCN2, either in melanoma cells or in the niche, impedes the ability of melanoma cells to invade. Specifically, loss of CCN2 in melanoma cells diminished their ability to invade through collagen in vitro, and loss of fibroblast-derived CCN2 decreased spontaneous metastases of melanoma cells from the skin to the lungs in vivo. Proliferation and tumor growth were not affected by loss of CCN2. CCN2-deficient B16(F10) cells showed reduced expression of the matricellular protein periostin; addition of recombinant periostin rescued the in vitro invasion defect of these cells. Immunohistochemical analysis of CCN2-deficient mice confirmed loss of periostin expression in the absence of CCN2. CCN2 and periostin mRNA levels are positively correlated with each other and with the stromal composition of human melanoma lesions but not BRAF mutations. Thus, CCN2 promotes invasion and metastasis via periostin and should be further evaluated as a possible therapeutic target for BRAF inhibitor-resistant melanoma.

Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models

Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high‐grade disease, Bacillus Calmette–Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell‐cycle‐regulated small subunit of ribonucleotide reductase (RRM2). The F4L‐deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor‐selective replication and cell killing. These F4L‐deleted VACVs replicated selectively in immune‐competent rat AY‐27 and xenografted human RT112‐luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY‐27 tumors by VACV treatment developed anti‐tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T‐lymphocyte assays. Finally, F4L‐deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L‐deleted VACVs, with application as a promising therapy for patients with BCG‐refractory cancers and immune dysregulation.

RUNX3 contributes to carboplatin resistance in epithelial ovarian cancer cells.

OBJECTIVE Resistance to platinum-based therapeutic agents represents a major hurdle in the treatment of epithelial ovarian cancer (EOC). There is an urgent need to better understand the underlying mechanisms. Here, we investigated the role of RUNX3 in carboplatin resistance in EOC cells. METHODS Expression of RUNX3 was determined in human EOC cell line A2780s (cisplatin-sensitive) and A2780cp (cisplatin-resistant), human ovarian surface epithelium (OSE) and primary EOC cells. The effects of RUNX3 expression on sensitivity to carboplatin were determined in A2780s and A2780cp cells using neutral red uptake and clonogenic assays. Carboplatin-induced apoptosis was determined by measuring cleaved PARP using Western blotting. The expression of cellular inhibitor of apoptosis protein-2 (cIAP2) and its regulation by RUNX3 were assessed by quantitative RT-PCR and Western blotting. RESULTS The expression of RUNX3 was elevated in A2780cp cells compared to A2780s cells and in EOC tissues from chemoresistant patients compared to those from chemosensitive patients. Overexpression of RUNX3 rendered A2780s cells more resistant to carboplatin, whereas inhibition of RUNX3 increased sensitivity to carboplatin in A2780cp cells. Inhibition of RUNX3 potentiated carboplatin-induced apoptosis in A2780cp cells as demonstrated by more pronounced PARP cleavage. Interestingly, the expression of cIAP2 was elevated in A2780cp cells compared to A2780s cells. Overexpression of RUNX3 increased cIAP2 expression in A2780s cells, whereas inhibition of RUNX3 decreased cIAP2 expression and potentiated carboplatin-induced decrease of cIAP2 in A2780cp cells. CONCLUSIONS RUNX3 contributes to carboplatin resistance in EOC cells and may hold promise as a therapeutic target to treat EOC and/or a biomarker to predict chemoresistance.

Investigating the utility of human melanoma cell lines as tumour models

Melanoma researchers utilize cell lines to model many tumour phenomena. It is thus important to understand similarities and differences between cell lines and the tumours that they represent, so that the optimal models can be chosen to answer specific research questions. Herein, we compared the transcriptomes of 42 melanoma cell lines to hundreds of tumours from The Cancer Genome Atlas and thousands of single melanoma cells. Tumour purity was accounted for using the ESTIMATE algorithm, so that differences likely resulting from non-tumour cells could be accounted for. In addition, UV mutational signatures and the expression of skin-associated genes were analyzed in order to identify the putative origin of various cell lines. We found the transcriptional and mutational characteristics of melanoma cell lines to mirror those of the tumours, with the exception of immune-associated transcripts, which were absent in cell culture. We also determined cell lines that highly or poorly recapitulate melanomas and have identified colon (COLO 741) and lung (COLO 699) cancer cell lines that may actually be melanoma. In summary, this study represents a comprehensive comparison of melanoma cell lines and tumours that can be used as a guide for researchers when selecting melanoma cell line models.

RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1

Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterized splice variant of RASSF1, RASSF1C, in cancer cell lines and primary tumors. We confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. The MOAP-1 promoter contains ~110 CpG sites, but was not found to be methylated in cancer cell lines when 19 predicted CpG sites were explored. Interestingly, MOAP-1 mRNA expression positively correlated with RASSF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A. We speculate that MOAP-1 and RASSF1A may be more intimately connected than originally thought, and the expression of both are warranted in experimental designs exploring the biology of the RASSF1A/MOAP-1 molecular pathway.