KRAS signaling enriched triple negative breast cancer is associated with favorable tumor immune microenvironment and better survival

Abstract

KRAS signaling is associated with cancer progression in several cancers. Upregulation of KRAS signaling is often seen in cancers that harbor high KRAS mutation rate, such as pancreatic cancer and non-small cell lung cancer (NSCLC). Less than 2% of breast cancers have KRAS mutation, however, the alteration of the effector signaling such as PI3K/AKT and MAPK pathways are well known. Mutated KRAS is known to function as immune suppressor in other cancers, but the role of KRAS signaling on tumor immune microenvironment (TIME) in breast cancer is not known. We hypothesize that the enrichment of KRAS signaling is associated with reduced patient survival as well as TIME in triple negative breast cancer (TNBC). Patient cohorts from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1903) and The Cancer Genome Atlas (TCGA; n = 982) were used. Higher expression of KRAS in breast cancer cell-lines (MCF7, BT474, and MDA-MB231) compared to MCF10A, which is a model of benign mammary cells was found. Both MEK and PI3K inhibitors suppressed MB231 cell proliferation in dose dependent manner. Gene Set Variant Analysis (GSVA) of the patient cohorts demonstrated two peaks by KRAS_SIGNALING_UP gene sets which were divided into KRAS-high and -low groups using median cutoff. There was no difference in KRAS mutation between KRAS-high and low. Despite its cell proliferation promoting role, KRAS-high patients demonstrated significantly better Disease-Free Survival and Overall Survival in triple negative breast cancer (TNBC). KRAS-high TNBC was associated with favorable tumor immune microenvironment with elevated B cells and CD8 T cells, monocytes, or M1 macrophage. It was associated with decreased CD4 central memory T-cells, but not Regulatory T-cells, or M2 macrophage detected by xCell. To elucidate the mechanism of this association, Gene Set Enrichment Analysis was performed. Inflammatory response, IL6/JAK-STAT3 signaling, and Interferon gamma response gene sets were enriched in KRAS-high TNBC patients in both METABRIC and TCGA cohorts. In agreement, cytolytic activity score, interferon gamma response score, and lymphocyte infiltrating signature score, were all significantly elevated in KRAS-high TNBC. In conclusion, we found that patients with enrichment of KRAS signaling gene sets were associated with inflammation and favorable tumor immune microenvironment as well as improved survival in TNBC.

Keywords: KRAS; KRAS signaling; METABRIC; TCGA; adaptive immune cells; breast cancer; innate immune cells; triple negative; tumor immune microenvironment.

Figure 1

KRAS signaling was functioning in the breast cancer cell lines. A. Up-regulation of KRAS expression in breast cancer cell lines when compared with MCF10A. Densitometric values were calculated for KRAS. B. Cell growth suppression after transfection of MB231 cells with MEK inhibitor or PI3K inhibitor at 72 H. Comb: PI3Ki 1 + MEKi 5 (μM). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

The patient distribution demonstrated bimodality with the scoring of gene set KRAS_SIGNALING_UP and higher scores in TNBC. A. Histogram of KRAS_SIGNALING_UP patients. Red line demonstrates median cutoff. B. The difference in scoring between estrogen receptor positive (ER+) and triple negative (TN) subgroups. C. The difference in scoring between KRAS wildtype and mutant.

Figure 3

KRAS-high TNBC demonstrated favorable prognostic outcome. Kaplan-Meier analysis of Disease-Free Survival (DFS) and Overall Survival (OS) in whole and TNBC.

Figure 4

KRAS-high group enriched immune related gene sets in both METABRIC and TCGA cohorts. A. The enriched gene sets of METABRC cohort. B. The enriched gene sets of TCGA cohort.

Figure 5

KRAS-high group demonstrated higher immune related scoring and cytolytic activity score (CYT) in whole and TN subgroup. A. IFN gamma Response Score of whole and TN subgroup with TCGA cohort. B. Lymphocyte Infiltrating Signature Score of whole and TN subgroup with TCGA cohort. C. Cytolytic activity score (CYT) of whole and TN subgroup with METABRIC cohort. D. CYT scoring of whole and TN subgroup with TCGA cohort.

Figure 6

Adaptive immune cells contribute to the shift of tumor immune microenvironment toward anti-cancer environment in KRAS-high TNBC. A. Adaptive immune cell composition within a tumor of METABRIC cohort. B. Adaptive immune cell composition within a tumor of TCGA cohort.

Figure 7

Innate immune cells contribute to the shift of tumor immune microenvironment toward anti-cancer environment in KRAS-high TNBC. A. Innate immune cell composition within a tumor of METABRIC cohort. B. Innate immune cell composition within a tumor of TCGA cohort.