Dual effect of KrasD12 knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy

Abstract

Activating mutations in the human KRAS proto-oncogene are acquired during the earliest stages of colorectal cancer development. If mutant KRAS is to be used as a target for therapy in colorectal cancer, tumor growth should depend on its continued presence. Here, we report that stable knockdown of Kras D12 in murine C26 colorectal cancer cells by RNA interference resulted in loss of transformed properties in vitro. The incidence of subcutaneous tumor formation was reduced by 60% and the lag time was increased sevenfold. Kras D12 -knockdown tumors grew noninvasively and did not cause morbidity. Remarkably, some of the Kras D12 -knockdown tumors regressed spontaneously, which rendered these mice resistant to parental C26 tumor growth. In immune-deficient hosts, the incidence of tumor formation by Kras D12 -knockdown cells was 100%. None of these tumors regressed spontaneously. We conclude that the reduced incidence of tumor formation by Kras D12 -knockdown cells is due to tumor cell clearance by the host immune system, but not to an intrinsic inability of these cells to grow out as tumors. Interestingly, Kras D12 knockdown resulted in increased production of interleukin 18 (Il-18), an immune-stimulatory cytokine that has been implicated in limiting colorectal tumor formation. Thus, mutant Kras D12 suppresses Il-18 production in colorectal tumor cells, which may contribute to evasion of the local immune system during tumor development.

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…The RAS family plays an important regulatory role in the processes of cell growth and differentiation, with KRAS acting as a molecular switch and participating in a number of cancer pathways such as that of colorectal cancer [48][49][50]. Studies have shown that 40-50% of human colorectal cancer is caused by the activity of KRAS protein expression, and inhibition of KRAS expression can inhibit tumor growth [51][52][53]. In recent years, a hypothesis that adherent A/E E. coli has a connection with colorectal carcinogenesis in humans has been put forth [54][55][56].…”

Transient infection of freshly isolated human colorectal tumor cells by reovirus T3D intermediate subviral particles

“…Directly following resection, the specimens were taken to the pathology department where a part of the tumor was excised for experimentation. The biopsy was mechanically dissociated using scalpels and vigorous trituration to yield small fragments (o1 mm 3 ) and single cells. In some cases, where indicated, the tissue fragments were incubated with thermolysin (0.05% in 10 mM Hepes pH 7.2, 37 1C, 1 h).…”

“…Recent evidence suggests that the sustained presence of oncogenic KRAS is required for maintenance of the transformed and malignant properties of several types of tumor cells. [1][2][3] This makes KRAS, or KRAS-activated signaling intermediates, attractive targets for therapeutic intervention. Reovirus T3D is an oncolytic virus that kills susceptible cells by inducing apoptosis.…”

Sensitization to Apoptosis Underlies Kras D12 -Dependent Oncolysis of Murine C26 Colorectal Carcinoma Cells by Reovirus T3D

“…C26 is an aggressive colorectal cancer cell line that contains constitutively activated Kras due to an activating point mutation in codon 12 (G12D) (22). The Nras and Hras genes in this cell line do not contain activating mutations.…”

“…As a control, we established cell lines transduced with the empty lentivirus pLL3.7 vector (C26-pLL). Efficient and specific knockdown of Kras was demonstrated by Western blot analysis for Kras and, as control, Nras (22). These cell lines were used to analyze the effect of Kras on C26 sensitivity to reovirus-induced oncolysis.…”