A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers with KRAS and BRAF mutations

Abstract

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

Implications of glutathione-S transferase P1 in MAPK signaling as a CRAF chaperone: In memory of Dr. Irving Listowsky

“…21) Similar GSTP1-related activation of target molecule, peroxiredoxin 6, of which the peroxidase activity protects cells against oxidant stress through GSH-dependent reduction of various hydroperoxides including phospholipid hydroperoxides to the corresponding water and alcohols, was also reported. 22) We also recently reported that GSTP1 binds to and activates CRAF and its downstream MAPK signaling in cancers with mutated KRAS or BRAF 9) these results will be described in following sections. Incidentally, there is a possibility that, although not related to the chaperone function, the above-mentioned thiol transferase activity of GSTP1 plays a role in MAPK signaling, because self-Sglutathionylation of GSTP1 resulted in interference with GSTP1/JNK complex formation.…”

“…To address this, we transduced siRNA GSTP1 and GSTP1 expression viral vectors into mKRAS cells and non-GSTP1expressing cancer cells, respectively, and confirmed a significant growth suppression of the former cells and growth enhancement of the latter cells, verifying the growth promoting activity of GSTP1 in mKRAS cells. 9) With regard to the mechanism of this growth promoting activity, however, the regulation of JNK activity by GSTP1 was not apparent as far as mKRAS cells were concerned, because the growth and death rates of mKRAS cells in the presence of JNK inhibitor were unchanged irrespective of siRNA GSTP1 treatment. 9) The chaperone activity of GSTP1 on CRAF, a signal component directly after mKRAS in the MAPK pathway, was unveiled because when we examined the MAPK signal components of mKRAS cells treated with siRNA GSTP1 or GSTP1 expression viral vector, decreased expression of p-CRAF/ CRAF/p-MEK/P-ERK or increased expression of the same respective signal components were observed.…”

“…9) With regard to the mechanism of this growth promoting activity, however, the regulation of JNK activity by GSTP1 was not apparent as far as mKRAS cells were concerned, because the growth and death rates of mKRAS cells in the presence of JNK inhibitor were unchanged irrespective of siRNA GSTP1 treatment. 9) The chaperone activity of GSTP1 on CRAF, a signal component directly after mKRAS in the MAPK pathway, was unveiled because when we examined the MAPK signal components of mKRAS cells treated with siRNA GSTP1 or GSTP1 expression viral vector, decreased expression of p-CRAF/ CRAF/p-MEK/P-ERK or increased expression of the same respective signal components were observed. 9) In a series of subsequent experiments, we showed that GSTP1 in mKRAS cells interacted with CRAF at its N-terminal domain to protect it from degradation by the ubiquitin-dependent proteasome pathway and to facilitate an essential step for signaling in the MAPK pathway involving the formation of RAF dimers; homodimer of CRAF and heterodimer of CRAF with BRAF (Fig.…”

“…4). 9) Because GSTP1, an end product of MAPK signaling, interacts with CRAF, an early signal component of the MAPK pathway, and enhances the signaling power, we designated this whole scheme as an "autocrine signal loop" (Fig. 4).…”

Pancreatic Cancer, Current Therapeutic Approaches and Possible Prospects

“…Однако, несмотря на разработку определенных ингибиторов специфических киназ, терапия не увенчалась значительным успехом. Более того, использование некоторых ингибиторов RAF вызывает ERK-зависимые эффекты обратной связи, которые, наоборот, вызывают усиление роста опухоли [42,43].…”