Oncol Rep. 2017 Aug;38(2):926-932.

Cetuximab promotes SN38 sensitivity via suppression of heat shock protein 27 in colorectal cancer cells with wild-type RAS

Takashi Ishida 1, Yoshiyuki Ishii 2, Masashi Tsuruta 1, Koji Okabayashi 1,

Shingo Akimoto 1, Kaoru Koishikawa 1, Hirotoshi Hasegawa 1 and Yuko Kitagawa 1

1) Department of Surgery, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582;

2) Department of Surgery, Kitasato University, Kitasato Institute Hospital, Shirokane 1-9-5, Minato-ku, Tokyo 108-8642, Japan. 

Correspondence should be addressed to Yoshiyuki Ishii, Department of Surgery, Kitasato University, Kitasato Institute Hospital, Shirokane 1-9-5, Minato-ku, Tokyo 108-8642, Japan, E-mail: yoshi6671@me.com.

 

Abstract:

Combination treatment with cetuximab and CPT-11 produces beneficial and synergistic effects in wild-type RAS metastatic colorectal cancer (mCRC) patients. However, the mechanism underlying this synergism is not yet understood. We examined whether cetuximab had a synergistic effect with CPT-11 and its active metabolite, SN38, and examined the molecular mechanism of the synergism between cetuximab and SN38 in CRC cells with various mutational status. We hypothesized that cetuximab promotes sensitivity to SN38 via suppression of heat shock protein 27 (HSP27), a protein involved in multidrug resistance through blocking the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which is associated with chemosensitivity. Four human CRC cell lines with different RAS and BRAF mutational status were used. Expression levels of HSP27 protein correlated with SN38 sensitivity in these cell lines (R=0.841, p=0.159). Exposure to cetuximab and various concentration of AG490, an inhibitor of JAK2, STAT3 and HSP27 protein levels, except in the KRAS G12V mutant line, SW620. A synergistic effect of cetuximab in combination with SN38 was observed in RAS and BRAF wild-type cells (here, Caco2), but not in the three other RAS– or BRAF mutated cell lines. These results indicate that cetuximab may promote sensitivity to SN38 via suppression of HSP27 through blocking the JAK/STAT pathway in Caco2 cells. The mutational status of numerous downstream effectors, such as RAS and BRAF, is important in mono- or combination therapy with cetuximab. In conclusion, cetuximab may promote SN38 sensitivity via suppression of HSP27, through blocking the JAK/STAT signaling pathway, and shows synergistic effects when combined with SN38 in wild-type RAS CRC cells.

PMID: 28656305

 

Supplement:

Cetuximab, a chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has markedly improved the prognosis in patients with metastatic colorectal cancer (mCRC) who harbor wild-type KRAS and BRAF in their tumors (1). Furthermore, new findings regarding RAS (NRAS) mutations can further assist in predicting the therapeutic effects of anti-EGFR antibody therapy (2). CPT-11, one of the major cytotoxic agents in mCRC, is converted into an active metabolite, SN38, which then acts as a topoisomerase I inhibitor. Several studies have shown that combination treatment with cetuximab and CPT-11 produces beneficial and synergistic effects in wild-type RAS mCRC (3). However, the mechanism underlying this synergism is not yet understood.

 

Mammalian heat shock proteins (HSPs) are known to be molecular chaperones in protein-protein interactions, acting as anti-apoptotic proteins and contribute to cell survival (4). HSPs have been classified into four major families, based on their molecular weights: HSP90, HSP70, HSP60 and ‘small’ HSPs (15-30 kDa), including HSP27 (5). Their expression can contribute to the malignant properties of cancer cells, including tumorigenicity, treatment resistance and apoptosis inhibition (5). Recently, HSP27 has been identified as a treatment target for several cancers, and clinical trials using an antisense oligonucleotide, OGX427, which inhibits HSP27 expression, have been performed in patients with prostate, bladder, ovarian, breast and non-small cell lung cancer, but not CRC (6). The therapy has been reported to be feasible and effective. In previous studies, we showed that HSP27 might contribute to resistance to 5-fluorouracil in CRC, in vitro and in vivo (7-9). It has also been reported that HSP27 expression is involved in resistance to CPT-11 and doxorubicin in vitro (10, 11). HSP27 is also believed to be involved in multidrug resistance (12). Thus, we considered that cetuximab might promote sensitivity to CPT-11 and SN38 via suppression of HSP27.

 

The janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway and, in particular, STAT3, which is a transcription factor, are known to have oncogenic potential (13). In CRC cells, activated STAT3 causes resistance to CPT-11 and inhibition of STAT3 strongly enhances the cytotoxic action of CPT-11 (14, 15). STAT3 activation in CRC patients is also associated with adverse clinical outcomes, supporting its potential roles as a prognostic biomarker and/or a therapeutic target (16). Furthermore, STAT3 has been reported to regulate HSP27 in breast epithelial cells (17).

 

We hypothesized that cetuximab might promote sensitivity to CPT-11 and SN38, via suppression of HSP27 through blocking of the JAK/STAT signaling pathway. At first, to assess whether cetuximab promoted SN38 sensitivity, we conducted the following experiment. CRC cells were treated with serial concentrations of SN38 (from 0.01 to 30 µg/mL) in the presence or absence of cetuximab. Combined drug effects were evaluated in RAS wild type CRC cells, Caco2. The addition of cetuximab (at a concentration causing ~20% inhibition) reduced the IC50 value of SN38 from 0.52 to 0.31 µg/mL (40% reduction) in Caco2 (Figure 1A; p = 0.046). Fractional effect-combination index (Fa-CI) plots of cetuximab and SN38 revealed synergistic interactions (Figure 1B; determined by calculated CI values < 1). These results suggest a synergistic effect of cetuximab and SN38 in Caco2 cells.

 

We elucidated that cetuximab promotes sensitivity to SN38 via suppression of HSP27, a protein involved in multidrug resistance through blocking JAK/STAT signaling pathway, which is associated with chemosensitivity including CPT-11, and shows synergistic effects when combined with SN38 in wild-type RAS CRC cells.

 

We consider that this study revealed a novel and important finding. Cetuximab may cause suppression of HSP27 through blocking the JAK/STAT signaling pathway in CRC cells, because AG490, an inhibitor of JAK2, caused a concentration-dependent decrease in the level of HSP27, similar to cetuximab. This suggests that HSP27 may be a downstream mediator of the JAK/STAT signaling pathway in CRC cells.

 

In order to further confirm this result, we conducted the experiment of stimulating each CRC cell with epidermal growth factor (EGF). Serum-starved CRC cells were stimulated with EGF (100 ng/ml) for 24hr. Western blot analyses showed that the expression of HSP27 and STAT3 were up-regulated by stimulation of EGF not only in RAS wild type CRC cells, Caco2, but also in RAS mutated cells, SW480 (Figure 2).

 

The lack of responsiveness to chemotherapy is an important problem of cancer treatment including CRC that has to be resolved. We consider that these findings, which revealed that cetuximab promotes sensitivity to SN38 by suppression of HSP27 via blocking JAK/STAT signaling pathway, may lead to new approches for mCRC treatment.

 

 

Figure 1. (A) Sensitivity to SN38 with or without cetuximab in Caco2 cells. The IC50 of SN38 decreased markedly when administered in combination with a low concentration of cetuximab (concentration to cause ~20% inhibition) for 48 h in Caco2 cells. (B) Fractional effect combination index (Fa-CI) plots of cetuximab and SN38 in Caco2 cells. Fa-CI plots revealed synergistic interactions (judged by calculated CI values < 1) in Caco2 cells. CI values were interpreted as follows: < 1, synergism; 1, additive; and > 1, antagonism. CI plots were prepared with CalcuSyn software (Biosoft, Cambridge, UK).

 

 

Figure 2. Serum starved CRC cells were stimulated with EGF (100ng/ml) for 24hr. Western blot studies showed that the expression of HSP27 and STAT3 were up-regulated by stimulation of EGF not only in RAS wild type CRC cells, Caco2,  but also in RAS mutated cells, SW480.

 

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