Background Cancer testis antigens (CTAs) are lucrative anti-cancer targets given their restricted expression patterns and known roles as mediators of cancer hallmarks, including cancer metabolism, proliferation, survival, and cell motility. Lactate dehydrogenase C (LDHC) is a CTA with upregulated expression in poor prognosis subtypes of breast cancer, however its tumorigenic role is less understood. We recently reported that silencing LDHC reduces breast cancer cell survival through a dysregulated DNA damage response, thus highlighting its potential as an anti-cancer target with limited off-target effects. This study aimed to explore the changes in the transcriptome of breast cancer cells and immune-related mediators upon in vitro LDHC targeting.
Materials and Methods We silenced LDHC expression in breast cancer cell lines and investigated the downstream effects on the tumor cell transcriptome. Differentially expressed genes were subjected to regulatory network analyses. We further assessed the secretory profile of cytokines and immune checkpoint expression in LDHC-silenced cells and used the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to determine the effect of the interaction between LDHC expression and cytotoxic T lymphocyte (CTL) infiltration in the METABRIC breast cancer cohort.
Results Network analysis to investigate the effects of silencing LDHC on the tumor cell transcriptome identified 47 up- and 55 down-regulated transcripts (2.0-fold change, adj p<0.05). Differentially expressed genes in the LDHC-silenced cells were particularly enriched in canonical pathways regulating cell cycle checkpoint control, BRCA1-mediated DNA damage response and NF-kb signaling in response to infection. Upstream regulator analyses revealed the altered expression profile was associated with mTOR (p=1.27e-5, z=2.242) and CASP3 (p=3.2e-4, z=2.250) pathways, which in the presence of LDHC are predicted to activate TP53, Myc, NF-KB complex, STAT1/3, PRKC, CDK2, FOXO3 and HIF-1a while inhibiting SMAD3, PTEN, ATM, IL18 and BCL2. Consequentially, the observed network-wide changes on LDHC silencing are predicted to negatively influence cellular growth and proliferation, cell migration and cell infiltration. The LDHC-associated network indicated a higher-level regulation by miR378a-3p (p=1.4e-7, z=-3.117), affecting the downstream mechanistic in LDHC-expressing cells. Interestingly, the miR378a causal network also indicated inhibition of the immune response in LDHC-positive cells. TIDE analysis indicated that high expression of LDHC in the METABRIC Her2 breast cancer cohort (TIDE score=1.97, p=0.049), and to a lesser extent in triple negative breast cancer (TIDE score=0.466, p=0.642), decreases the beneficial effect between CTLs and overall survival observed in LDHC Low tumors. Concurrently, LDHC-silenced cells displayed a pro-inflammatory gene expression and cytokine profile and down-regulated the expression of PD-L1 and Gal-9 immune checkpoints.
Conclusions Our findings provide an indication of potential CTL dysfunction in breast tumors with high LDHC expression and suggests that therapeutic targeting of LDHC may inhibit tumor growth while releasing the anti-tumor immune response in breast cancer.
Disclosure Information A. Naik: None. J. Decock: None.