Interleukin 6 Alters Localization of hMSH3, Leading to DNA Mismatch Repair Defects in Colorectal Cancer Cells

Abstract

Background & Aims Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair (MMR) defect in colorectal cancers, observed in ~60% of specimens. This acquired genotype correlates with metastasis and poor outcome of patients, and is associated with intra-epithelial inflammation and heterogenous nuclear levels of the MMR protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. Methods We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and ELISA; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble sgp130Fc fragments, and the STAT3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent non-tumor tissues. Results Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other MMR proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 that EMAST-negative tumors. Conclusions IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

<i>Fusobacterium nucleatum</i> Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations

“…As mentioned above, cumulative evidence supports the idea that the etiology of MSI-L/EMAST formation in cancer genome is the inflammation-induced loss of nuclear MSH3 in dividing cells (10,37,39). Therefore, it is reasonable to speculate that a sub-group of MSI-L/EMAST CRC may be associated with pathogenic infections of microbiota including Fusobacterium .…”

“…These results suggest that some immunological and inflammatory responses are active in EMAST CRC. Later, Tseng-Rogenski et al demonstrated that in several cancer cell lines (37,39) inflammatory factors including oxidative stress (hydrogen peroxide), interleukin-6 (IL6) and prostaglandin E 2 (PGE 2 ) induce displacement of MSH3 from the nucleus to the cytoplasm, whereas the other MMR proteins do not displace. Repeated treatment of microsatellite stable colon cancer cell lines with IL6 induced EMAST.…”

Microsatellite Alterations With Allelic Loss at 9p24.2 Signify Less-Aggressive Colorectal Cancer Metastasis

“…Recent studies suggest that E/L in non-MSI-H CRC may be caused by loss of MSH3 in a cancer cell due to the effects of tumor micro-environmental factors, such as hypoxia and/or inflammation, rather than genetic causes (17, 21, 28, 29, 30). Previously, we demonstrated that E/L in Stage II/III primary CRC is associated with a high risk for recurrent distant metastasis (21).…”

Prognostic Impact of IL6 Genetic Variants in Patients with Metastatic Colorectal Cancer Treated with Bevacizumab-Based Chemotherapy

“…Importantly, we were not able to correlate IL-6 polymorphisms with serum or intratumoral IL-6 expression levels which may lend insight on mechanisms of bevacizumab resistance. Nor were we able to determine interactions between microsatellite instability 8,49 , NSAID use, or inflammatory bowel disease, all of which may be affect the influence of IL-6 polymorphisms on outcomes. Functional studies are certainly needed to determine the significance of each examined genetic variant.…”

“…As initiators of pathologic inflammation, IL-6 and its downstream effector, STAT3 (signal transducer and activator of transcription-3), promote colorectal cancer (CRC) development 3,4 , invasion and metastasis 5–7 , by imposing genetic alterations in tumor cells 3,8 and facilitating immune tolerance within the microenvironment 6,9,10 . In colitis-associated cancer in vivo models, IL-6 enhances tumor cell proliferation and protects normal and malignant intestinal epithelial cells from apoptosis in a STAT3 dependent fashion.…”