Background: Previously we identified a DNA damage response–deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher’s exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response–proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response–proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle–specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint–based therapies.

Cathepsin S is a member of the cysteine cathepsin protease family. It is a lysosomal protease which can promote degradation of damaged or unwanted proteins in the endo-lysosomal pathway. Additionally, it has more specific roles such as MHC class II antigen presentation, where it is important in the degradation of the invariant chain. Unsurprisingly, mis-regulation has implicated cathepsin S in a variety of pathological processes including arthritis, cancer, and cardiovascular disease, where it becomes secreted and can act on extracellular substrates. In comparison to many other cysteine cathepsin family members, cathepsin S has uniquely restricted tissue expression and is more stable at a neutral pH, which supports its involvement and importance in localised disease microenvironments. In this review, we examine the known involvement of cathepsin S in disease, particularly with respect to recent work indicating its role in mediating pain, diabetes, and cystic fibrosis. We provide an overview of current literature with regards cathepsin S as a therapeutic target, as well as its role and potential as a predictive diagnostic and/or prognostic marker in these diseases.

Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the "resistant" population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity.

Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.

BackgroundCathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.MethodsValidation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true Ki values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.ResultsWe confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.ConclusionsIn summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0513-7) contains supplementary material, which is available to authorized users.