SUMMARY Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

IntroductionPodocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.MethodsWe silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice.ResultsAlthough deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis.ConclusionsWe show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0562-7) contains supplementary material, which is available to authorized users.

High-throughput transcriptome sequencing allows identification of cancer-related changes that occur at the stages of transcription, pre-messenger RNA (mRNA), and splicing. In the current study, we devised a pipeline to predict novel alternative splicing (AS) variants from high-throughput transcriptome sequencing data and applied it to large sets of tumor transcriptomes from The Cancer Genome Atlas (TCGA). We identified two novel tumor-associated splice variants of matriptase, a known cancer-associated gene, in the transcriptome data from epithelial-derived tumors but not normal tissue. Most notably, these variants were found in 69% of lung squamous cell carcinoma (LUSC) samples studied. We confirmed the expression of matriptase AS transcripts using quantitative reverse transcription PCR (qRT-PCR) in an orthogonal panel of tumor tissues and cell lines. Furthermore, flow cytometric analysis confirmed surface expression of matriptase splice variants in chinese hamster ovary (CHO) cells transiently transfected with cDNA encoding the novel transcripts. Our findings further implicate matriptase in contributing to oncogenic processes and suggest potential novel therapeutic uses for matriptase splice variants.

Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralization of the changes in gene expression has been shown to be attractive targets for the development of new anti-cancer drugs and therapeutic strategies. New approaches such as antibody drug conjugates (ADCs) also target differentially expressed genes as a mean to recognize tumor cells to selectively deliver toxins to a tumor. In the past decade, the global analysis of gene expression in human cancers have led to the development of a number of potential new biomarkers. For instance, mesothelin (MSLN) was identified as an over-expressed gene in pancreatic cancer and later was proved to be a useful diagnostic marker and so a therapeutic target. Large-scale gene expression analysis, using techniques such as RNA sequencing, provides a powerful tool to identify genes involved in human cancers. In this study, with the ultimate goal being to identify potential novel targets for cancer immunotherapy, we conducted a pan-cancer differential expression analysis in RNA sequencing data from more than 5,000 patients with 25 different cancer types generated by The Cancer Genome Atlas (TCGA). We identified differentially expressed genes (present in at least 5% of samples in a tumor type) in comparison to a large compendium of normal transcriptomes (more than 650 samples, including 30 tissue types) gathered from Genotype-Tissue Expression (GTEx), illumina BodyMap project 2.0, TCGA, and an in-house database. In total, we identified 892 putative tumor-associated differentially expressed genes. In order to further identify novel candidate genes and rank them based on their antigenic potential, we performed an extensive literature search and systematic review to collect the characteristics of an ideal tumor antigen (TA). We developed an Analytic Hierarchy Process (AHP) model - a multiple-criteria decision-making solution - to depict antigen properties for ranking and prioritizing the tumor-associated differentially expressed genes. Our model recognizes the known tumor antigens (such as CA9, Nectin-4, FN1, MSLN and MUC16, which are currently in clinic or pre-clinical studies) in the top 25 of the ranked list. We are currently validating the top-ranked novel antigens in an orthogonal panel of tumor and normal tissues and cell lines using PCR. Note: This abstract was not presented at the meeting. Citation Format: Daryanaz Dargahi, Richard D. Swayze, Leanna Yee, Peter J. Bergqvist, Bradley J. Hedberg, Alireza Heravi-Moussavi, Edie M. Dullaghan, Ryan Dercho, Christopher Bond, Jianghong An, John S. Babcook, Steven JM Jones. Pan-cancer identification and prioritization of cancer-associated differentially expressed genes: A biomarker discovery application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2187. doi:10.1158/1538-7445.AM2015-2187