Intercellular Transfer of Oncogenic KRAS via Tunneling Nanotubes Introduces Intracellular Mutational Heterogeneity in Colon Cancer Cells.

Desir Desir,Shetty Shetty,Starr Starr,Lou Lou,Wong Wong,Nissley Nissley,Zhai Zhai,Manova-Todorova Manova-Todorova,Turbyville Turbyville,Chen Chen,Romin Romin,Steer Steer,Clark Clark,Subramanian Subramanian

doi:10.3390/cancers11070892

Abstract

Mutated forms of the RAS oncogene drive 30% of all cancers, but they cannot be targeted therapeutically using currently available drugs. The molecular and cellular mechanisms that create a heterogenous tumor environment harboring both mutant and wild-type RAS have not been elucidated. In this study, we examined horizontal transfer of mutant KRAS between colorectal cancer (CRC) cells via a direct form of cell-to-cell communication called tunneling nanotubes (TNTs). TNT formation was significantly higher in CRC cell lines expressing mutant KRAS than CRC cell lines expressing wild-type RAS; this effect was most pronounced in metastatic CRC cell lines with both mutant KRAS and deficiency in mismatch repair proteins. Using inverted and confocal fluorescence time-lapse and fluorescence recovery after photobleaching (FRAP)-based microscopy, we observed GFP-tagged mutant KRASG12D protein trafficking between CRC cells through TNTs within a span of seconds to several minutes. Notably, acquisition of mutant KRAS increased Extracellular Signal-regulated Kinase (ERK) phosphorylation and upregulated tunneling nanotube formation in recipient wildtype CRC cells. In conclusion, these findings suggest that intercellular horizontal transfer of RAS can occur by TNTs. We propose that intercellular transfer of mutant RAS can potentially induce intratumoral heterogeneity and result in a more invasive phenotype in recipient cells.

Highlights

RAS is a ubiquitous oncogene in cancers and is highly active and prevalent in both pancreatic (90–95% KRAS mutations) and colorectal cancers (CRC) (35–40%)
We have previously found that the rate of tunneling nanotubes (TNTs) formation is heterogeneous and variable even among cancer types of similar tissue of origin
We hypothesized that colon carcinoma cells form TNTs at rates that vary based on KRAS status and site of origin (Table 1)

Summary

Introduction

RAS is a ubiquitous oncogene in cancers and is highly active and prevalent in both pancreatic (90–95% KRAS mutations) and colorectal cancers (CRC) (35–40%). There is increasing evidence that mutated versions of KRAS lead to the development of chemoresistance and that subclones of mutated KRAS are present at the time of diagnosis of CRC even in tumors that are initially identified as wild-type (wt) for KRAS [7]. Intratumoral heterogeneity of KRAS, in which multiple alleles of the oncogene exist within an individual tumor, can lead to the misdiagnosis of tumors as wild-type RAS. Such tumors, when treated with monoclonal antibody targeting the epidermal growth factor receptor (EGFR), will eventually develop resistance to anti-EGFR therapy, a characteristic most prominent in colorectal cancer [17,18]. We hypothesized that tunneling nanotubes (TNTs) provide an additional mechanism of intercellular communication of oncogenic

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Discussion

Conclusion