The immune co-inhibitory receptors lymphocyte activation gene-3 (LAG3) and programmed cell death 1 (PD1) synergistically contribute to autoimmunity and tumor evasion. Here we demonstrate how they collaborate and interact to regulate T cell function. We first show that LAG3 and PD1 are co-expressed on both OVA-specific and non-specific T cells infiltrating murine ovarian tumors. Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed tumor growth. LAG3 and PD1 co-localized in activated CD8+ T cells in vitro at the trans-Golgi vesicles, early/recycling endosomal compartments, lysosomes, and microtubule organizing center. Importantly, LAG3 and PD1 cluster with pLck at the immunological synapse. Reciprocal immunoprecipitation of T cell extracts revealed physical interaction between LAG3 and PD1. Mutational analyses indicate that the cytoplasmic domain of LAG3 is not absolutely required for its association with PD1, while the ITIM and ITSM of PD1 are necessary for its association with LAG3. Finally, LAG3 protein also associates with the Src-homology-2 domain-containing phosphatases (SHP1/2) which are known to be recruited by PD1 during T cell signaling. Our data indicate that the association of LAG3 with PD1 contributes to their rapid trafficking to the immunological synapse, leading to a synergistic inhibitory effect on T cell signaling.

Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. In the present study we have tested whether serine-arginine protein kinase 1 (SRPK1), a major regulator of splicing factors, is involved in ovarian cancer progression and plays a role in chemo-sensitivity. By Western blot analyses, SRPK1 protein was found to be overexpressed in 4 out of 6 ovarian cancer cell lines as compared with an immortalized ovarian surface epithelial cell line; and in 55% of ovarian tumor samples as compared with non-neoplastic ovarian tissue samples. Reduction of SRPK1 expression using small interfering RNA (siRNA) encoding small hairpin RNA in ovarian cancer cells led to (i) reduced cell proliferation rate, slower cell cycle progression and compromised anchorage-independent growth and migration ability in vitro, (ii) decreased level of phosphorylation of multiple serine-arginine proteins, and P44/42MAPK and AKT proteins, and (iii) enhanced sensitivity to cisplatin. Together, these results suggest that elevated SRPK1 expression may play a role in ovarian tumorigenesis and SRPK1 may be a potential target for ovarian cancer therapy.

ST6Gal I (β-galactoside α2,6-sialyltransferase, ST6N) elaborates the ubiquitously expressed α2,6-sialyl linkage. A number of ST6Gal I mRNA isoforms, differing only in their 5′-UT regions, is transcribed from a single mouse gene, Siat1. In B-lineage cells, α2,6-sialic acid serves as extracellular ligand for CD22, a participant in cell activation via an intracellular signaling network of tyrosine kinases and SHP phosphastase. Activation and terminal differentiation of mature B cells into plasma cells is accompanied by the appearance of at least four distinct ST6Gal I mRNA isoforms. Resting splenic B-lymphocytes isolated from 8-12 wk C56Bl/6 mice expressed almost exclusively the Exons Q+O-containing form, which is the likely homolog to the previously documented human Y+Z and rat -1+0 forms. In vitro activation using recombinant CD40-ligand and conditioned media from T-helper cells resulted in a 2-to 3-fold elevation of overall ST6Gal I mRNA abundance by Day 3. This coincided with repression of the Q+O form, and appearance of three new isoforms containing 5′-untranslated sequences X 1 , X 2 , or X 3 . The X 1 form persisted through Day 10, when the transition of B cells to plasma cells was completed as evidence by disappearance of CD22 mRNA. In contrast, the X 2 form only transiently appeared at Day 3 and declined to barely detectable levels by Day 7. Expression of the X 3 form, a minor mRNA form, paralleled the X 2 form. The divergent 5′-UT exons are dispersed over 69 kb of linear genomic space of Siat1. Mutually exclusive utilization of these 5′-UT exons in transcripts predicts separate and distinct promoter regulatory regions for each mRNA isoform.

The sialyltransferase ST6Gal mediates the biosynthetic addition of sialic acid, via an alpha2,6 linkage, to the nonreducing end of terminal lactosamine structures. Transcription of the murine ST6Gal gene, Siat1, is regulated by the selective use of multiple promoters in a tissue- and development-specific manner. Here we report that Siat1 mRNA expression is dramatically elevated in lactating (relative to virgin) mouse mammary gland. The predominant ST6Gal mRNA species expressed in lactating mammary gland is a heretofore undocumented isoform containing a unique 5'-untranslated region originating from the mouse Siat1 genetic region, now defined as Exon L, residing 549-bp 5' of the previously characterized Exon X(2). Thus, the novel ST6Gal mRNA form initiates transcription from the region designated as p4 and incorporates the unique sequence from Exon L in 5'-juxtaposition to commonly shared sequences encoded on Exon I to Exon VI. In contrast, cells derived from virgin mammary tissue expressed only the housekeeping mRNA form derived from p3, with Exon O sequence preceding Exons I-VI. The Exon L-containing, p4 class of mRNA was also not detected in a survey of eight other mouse tissues. Previous reports have indicated a strong correlation between mammary cancers and elevated ST6Gal expression in rats and in human patients. However, we uncovered neither elevated expression of ST6Gal mRNA nor appearance of p4 class in mouse breast carcinomas experimentally induced by transformation with the polyoma-middle T oncogene. A number of established breast carcinoma cell lines were also examined, with ST6Gal mRNA and activity generally low. Moreover, with the exception of the Shionogi cell line, p4 class of ST6Gal mRNA was not expressed in any of the mouse breast carcinoma specimens examined. Taken together, our data indicate that murine ST6Gal induction during lactation is achieved by de novo recruitment of a normally silent promoter. Furthermore, the data provide no support for elevated Siat1 expression on the mRNA level in association with murine mammary gland carcinogenesis. With the single exception of the Shionogi cell line, the p3 class remains the predominant ST6Gal mRNA expressed in all other murine mammary carcinoma cells examined.

An association between genetic variants in the vitamin D receptor ( VDR ) gene and epithelial ovarian cancer (EOC) was previously reported in women of African ancestry (AA). We sought to examine associations between genetic variants in VDR and additional genes from vitamin D biosynthesis and pathway targets ( EGFR, UGT1A, UGT2A1/2, UGT2B, CYP3A4/5 , CYP2R1 , CYP27B1 , CYP24A1 , CYP11A1 , and GC ). Genotyping was performed using the custom‐designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set in 755 EOC cases, including 537 high‐grade serous (HGSOC), and 1,235 controls. All subjects are of African ancestry (AA). Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (CI). We further evaluated statistical significance of selected SNPs using the Bayesian False Discovery Probability (BFDP). A significant association with EOC was identified in the UGT2A1/2 region for the SNP rs10017134 (per allele OR = 1.4, 95% CI = 1.2‐1.7, P = 1.2 × 10 −6 , BFDP = 0.02); and an association with HGSOC was identified in the EGFR region for the SNP rs114972508 (per allele OR = 2.3, 95% CI = 1.6‐3.4, P = 1.6 × 10 −5 , BFDP = 0.29) and in the UGT2A1/2 region again for rs1017134 (per allele OR = 1.4, 95% CI = 1.2‐1.7, P = 2.3 × 10 −5 , BFDP = 0.23). Genetic variants in the EGFR and UGT2A1/2 may increase susceptibility of EOC in AA women. Future studies to validate these findings are warranted. Alterations in EGFR and UGT2A1/2 could perturb enzyme efficacy, proliferation in ovaries, impact and mark susceptibility to EOC.