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. 2019 Jan 22;38(1):28.
doi: 10.1186/s13046-018-1019-5.

JAK/Stat5-mediated subtype-specific lymphocyte antigen 6 complex, locus G6D (LY6G6D) expression drives mismatch repair proficient colorectal cancer

Guido Giordano  1   2 , Pietro Parcesepe  3 , Mario Rosario D'Andrea  4 , Luigi Coppola  4 , Tania Di Raimo  4 , Andrea Remo  5 , Erminia Manfrin  3 , Claudia Fiorini  3 , Aldo Scarpa  3 , Carla Azzurra Amoreo  6 , Fabiana Conciatori  7 , Michele Milella  7 , Francesca Pia Caruso  8   9 , Luigi Cerulo  8   9 , Almudena Porras  10   11 , Massimo Pancione  12   13
Affiliations

JAK/Stat5-mediated subtype-specific lymphocyte antigen 6 complex, locus G6D (LY6G6D) expression drives mismatch repair proficient colorectal cancer

Guido Giordano et al. J Exp Clin Cancer Res. .

Abstract

Background: Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically "cold" tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear.

Methods: We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies.

Results: We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC.

Conclusions: Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.

Keywords: Colorectal cancer; Immune resistance; LY6G6D; Microsatellite-stable.

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Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the San Filippo Neri Hospital, Rome, Italy and has been performed in accordance with the ethical guidelines as reported in the 1964 Helsinki Declaration and its later amendments.

Consent for publication

Not applicable.

Competing of interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characterization of LY6G6D and FUT4/CD15 expression. a The work flow on the left shows cancer cell line transcriptomic samples that were retrieved from NCBI (Barretina J et al. 2102) and interrogated for differentially expressed genes of known immune-related genes from ImmPort collection. Right, unsupervised hierarchical cluster of cancer cell lines (n = 604) shows a gene signature enriched in colorectal cancer. Enlarged image shows two genes LY6G6D and FUT4/CD15 within the cluster that are upregulated in Microsatellite stable (MSS) but not in microsatellite instable (MSI) colon cancer cells categorized for mutational load and copy number variations (CNVs). b Quantification of CD15 and LY6G6D mRNA in patient-matched tumor-normal mucosa extracted from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data sets. Scatter plot in which each circle represents mRNA levels in each tumor sample, horizontal line is the mean value. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by Mann–Whitney U test. c Heat map of log-transformed odds ratios of a set of immune-related genes for two different molecular phenotypes MSI vs MSS. On the left, quantification of LY6G6D mRNA by a box plot in CRCs classified as CIN high or low based on a weighted genome integrity index (see Methods). *P ≤ 0.05; t test Welch-corrected. d Enrichment map network of statistically significant gene interactions. Nodes represent gene hub and lines their connectivity. Node size is proportional to number of line with arrows. Heat map of differentially expressed genes within JAK/STAT and MAPK signaling according to MSI-H, MSI-L, MSS subtypes. Shown are groups with high relative expression (hi, red) versus the low relative expression (lo, blue) at the optimum value cutoff
Fig. 2
Fig. 2
Intra-tumoral immunophenotypes marked by LY6G6D and FUT4/CD15. a On the top, unsupervised hierarchical cluster of 232 CRC samples (dataset: GSE17536–37) using cell-specific immune-signatures categorized patients into four groups, with distinct cell immune associated gene expression. Data are obtained using the Euclidean distance and Ward linkage method on the matrix of the enrichment scores calculated through ssGSEA. Top tracks represents the expression profile of known immune inhibitory molecules, together with LY6G6D and CD15/FUT4 genes. On the bottom, boxplots of LY6G6D gene expression in each cluster. b Dot plot representing the mean enrichment scores of each immune cell type in any cluster. Color scale represents the positive (red) and negative (blue) enrichment score; dot size indicates the strength of the association. c representative western blot images and quantification of LY6G6D and CD15 expression from CRC samples and matched normal mucosa (n = 12) relative to β-actin used as loading. Data are mean ± standard error of the mean (s.e.m); (n = 3 biological replicates, P* < 0.05, ***P < 0.001, two-tailed Student’s t-test. Low, LY6G6D and CD15 IHC in normal mucosae and tumor specimens; Scale bar, 100 μm. Enlarged is the staining in both malignant cells (T) and stromal (S) immune cells. d Correlation between LY6G6D+ cells, CD8 T-lymphocytes and CD86 staining in CRC samples (five replicates counts, cells mm− 2). Double immunofluorescence from paraffin embedded sections co-stained with antibodies against CD4 (red) and FOXP3 (red) or LY6G6D (green). Scale bar, 50 μm and 20 μm, respectively
Fig. 3
Fig. 3
Immune inhibitory molecules in MSI and MSS tumours. a quantification of infiltrating LY6G6D positive cells expressed as mean of five replicates counts, cells mm− 2) in normal mucosa and CRC samples. Correlation between LY6G6D+ cells, p-STAT5 staining in CRC samples (five replicates counts, cells mm− 2). b Examples of MSI and MSS CRC stained by immunohistochemistry against MLH1, LY6G6D, PDL1 and PD1. T, Tumour, S, stromal compartment. Scale bar, 50 μm. c Quantification of stromal infiltration and staining of malignant cells by immunohistochemistry for LY6G6D, PDL1 and PD1. P* < 0.05, P** < 0.01, ***P < 0.001, by the Chi-squared test. d Kaplan–Meier curve showing the time to disease progression in relation to LY6G6D and CD15 status (n = 187); The p-value by log-rank test. Response to treatment according to LY6G6D IHC in primary metastatic tumors (n = 83) subdivided for complete (CR); partial (PR) responses; stable disease (SD) and progression disease (PD); P* < 0.05, P** < 0.01, ***P < 0.001, by the Chi-squared test
Fig. 4
Fig. 4
Response to JAK/STAT and MEK inhibitors in CRC molecular subtypes. a Heat map showing mutation/expression of JAK/STAT genes in relation to mutation load, LY6G6D and CD15/FUT4 expression in CRC cancer cell lines (n = 38). b A chemo-immune-sensitizer approach targeting LY6G6D and CD15/FUT4 by JAK/STAT and MEK inhibitors. Right, Log10 IC50 values for treatment of MSI and MSS CRC cell lines with ruxolitinib (JAK/STATi) and trametinib (MEKi) extracted from the Genomics of Drug Sensitivity in Cancer project. c RKO (MSI-H) and SW620 (MSS) stained with LY6G6D (green) and CD15 (red). Bottom right, basal activation of stat1, stat3, stat5 in a panel of CRC cell lines. Down left, western blotting showing expression of P-STAT5, STAT5 P-ERK1/2, ERK1/2 and LY6G6D. Down right, quantification of P-STAT5, P-ERK1/2 and LY6G6D relative to β-actin. d Cells were treated with different concentrations of momelotinib (range, 1 nM to 1 mM for 96 h) and evaluated for proliferation by MTT staining. Right, boxplot of log10 IC50 values for treatment of five CRC cell lines (RKO,HT29, SW480, SW620, HCT116) with ruxolitinib vs momelotinib. Results are representative of three biological replicates. P-value by two-tailed Student’s. P* < 0.05, **P < 0.01
Fig. 5
Fig. 5
MSS CRC cell lines are highly sensitive to STAT5/MEK inhibitors. a MSS BRAF(V600E), KRAS mutant and b MSI BRAF(V600E), KRAS mutant CRC cells were seeded at low confluence and treated with increasing concentrations (lower than IC50 values) of momelotinib, trametinib or in combination (comb) twice a week. Viability was assessed by a colony formation assay. Cells were fixed, stained, and photographed after 10 days of culture. For each cell line, in the low panel the percent of cell growth inhibition determined by treatment is shown. Results represent three separate experiments, each performed in triplicate. P-value by two-tailed Student’s (related to untreated vehicle control) are shown, P* < 0.05, **P < 0.01, ***P < 0.001, NS, non significant. c representative immunoblot of phosphorylated STAT5 and ERK1/2 compared to LY6G6D following treatment with momelotinib, trametinib or combination. Bottom right, quantification to β-actin. Low left, viability of HCT116 cell lines (KRAS mutant), and its derivative HKE-3 KRAS wild type (KRASWT) to momelotinib, trametinib or their combination assessed by colony formation assay. Low on the right, quantification of LY6G6D and FUT4 mRNA by RT-PCR analysis following drug treatments. ***P < 0.001 by Mann–Whitney U test. d Illustration of immune suppressive pathway mediated by LY6G6D and CD15, which could predict response to JAK- and MAPK-directed therapies in microsatellite stable CRC
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