NRAS destines tumor cells to the lungs

Abstract

The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross‐examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1‐ and Cxcr2‐deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin‐8‐related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS‐mutant, chemokine‐expressing circulating tumor cells target the CXCR1‐expressing lung vasculature and recruit CXCR2‐expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS‐driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL‐8‐related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

An in vivo inflammatory loop potentiates KRAS blockade

“…NCI-H358, NCI-H358M, NCI-H460, NCI-H520, NCI-H1299, NCI-H1944, NCI-H3122 (referred to hereafter omitting NCI), EKVX, A549, LLC, B16F10, and PANO2 cell lines were from the National Cancer Institute (Frederick, MD); MC38 cells were a gift from Dr. Timothy S. Blackwell (Vanderbilt University, Nashville, TN) and AE17 cells from Dr. YC Gary Lee (University of Western Australia, Perth, Australia) [13–15]. FULA1 ( FVB urethane-induced lung adenocarcinoma 1) and CULA ( C57BL/6 urethane-induced lung adenocarcinoma) cell lines were isolated from the lungs of FVB and C57BL/6 mice, respectively, harboring primary lung adenocarcinomas induced by urethane [13,16,17].…”

“…FVB/NJ ( FVB ; #001800), C57BL/6J ( C57BL/6 ; #000664), B6.129P2- Cxcr1 tm1Dgen/J ( Cxcr1 -/- ; #005820) [15], B6.129S4- Ccr2 tm1Ifc/J ( Ccr2 -/- ; #004999) [16], B6.129S2(C)- Cxcr2 tm1Mwm/J ( Cxcr2 +/- ; #006848) [15], and B6(Cg)- Rag2 tm1.1Cgn /J ( Rag2 -/- ; #008449) mice were from the Jackson Laboratory (Bar Harbor, ME) and Il1b tm1Yiw mice ( Il1b -/- ; MGI #2157396) [18] were a kind gift from Dr. Yoichiro Iwakura (Tokyo University of Sciences, Japan). All mice were bred at the Center for Animal Models of Disease of the University of Patras.…”

“…Il1r1/IL1R1 and Gapdh/GAPDH qPCR was performed using specific primers (Supplementary Table S2) and Lightcycler 480 Sybr Green I Master (Roche; Mannheim, Germany) in a Lightcycler 480 II (Roche Diagnostics). Ct values from triplicate reactions were analyzed with the 2 -ΔCT method as detailed elsewhere [15]. mRNA abundance was determined relative to Gapdh/GAPDH and is given as 2 -ΔCT = 2 -(Ct of Il1r1/IL1R1 )-(Ct of Gapdh/GAPDH ) .…”

“…mRNA abundance was determined relative to Gapdh/GAPDH and is given as 2 -ΔCT = 2 -(Ct of Il1r1/IL1R1 )-(Ct of Gapdh/GAPDH ) . Mouse microarrays were done as described elsewhere [13–15]. Briefly, triplicate cultures of 10 6 cells were subjected to RNA extraction as above, 5 μg of pooled total RNA were tested for RNA quality on an ABI2000 Bioanalyzer (Agilent; Santa Clara, CA), labelled, and hybridized to GeneChip Mouse Gene 2.0 ST arrays (Affymetrix; Santa Clara, CA).…”

“…For adoptive bone marrow transplants (BMT), bone marrow cells were flushed from both femurs and tibias of wild-type ( WT ) or Ccr2 -/- mice (all back-crossed >F12 to the FVB background) using fully supplemented DMEM. Ccr2 -/- mice (all FVB ) received ten million intravenous (iv) bone marrow cells from WT or Ccr2 -/- mice 12 hours after total-body irradiation (900 Rad), as described elsewhere [14,15,18]. One mouse in each experiment was not engrafted and was observed till moribund on days 5-15 post-irradiation.…”