We conducted this study to examine prognostic significance of p-AMPK (phosphorylated AMP-activated protein kinase; p-PRKA) expression in a large cohort of colorectal cancers. To our best knowledge, no previous study has examined its prognostic role in human colorectal cancer. Considering a pivotal role of AMPK as a regulator of cellular metabolism and the relationship of AMPK with the MAPK3/1 (ERK1/2) pathway and other signaling pathways, we hypothesised that cellular AMPK might interact with MAPK3/1 to modify tumour behaviour. Notably, we found that the prognostic effect of p-AMPK expression differed according to p-MAPK3/1 status. hosphorylated AMPK expression was associated with superior survival among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases. Our results support an interaction between the AMPK and MAPK3/1 pathways in colorectal cancer cells to modify tumour behaviour.
Examining molecular changes or prognostic factors is important in cancer research (Fluge et al, 2009
; Gaber et al, 2009
; Jubb et al, 2009
; Rasheed et al, 2009
; Kontos et al, 2010
; Rego et al, 2010
; Zlobec et al, 2010
). Accumulating evidence suggests that AMPK acts as a tumour suppressor. STK11 (LKB1) has been identified as an upstream activator of AMPK (Shackelford and Shaw, 2009
), and TSC2, which is a negative regulator of MTOR, is a downstream effector of AMPK (Inoki and Guan, 2009
). Experimental studies have shown that AMPK activation inhibits cancer cell proliferation and growth (Buzzai et al, 2007
; Zakikhani et al, 2008
). In a study using 354 breast cancers (Hadad et al, 2009
), p-AMPK expression was not significantly associated with prognosis, but modifying effect of MAPK3/1 was not examined. To our knowledge, no previous study has examined the prognostic role of AMPK in colorectal cancer.
Considering experimental data on the link between the STK11 (LKB1)-AMPK and MAPK3/1 pathways, the modifying effect of MAPK3/1 on AMPK may not be surprising. In colon cancer cells, AMPK potentially inhibits the MAPK3/1 pathway; inhibition of AMPK by expressing a dominant-negative form potentiates MAPK3/1 activation under glucose deprivation (Kim et al, 2010
). Selenium, an essential trace element, blocks the carcinogenic agent-induced MAPK3/1 activation via AMPK (Hwang et al, 2006
). AMP-activated protein kinase is rapidly activated by cisplatin and suppresses an apoptotic signal via MAPK3/1 in colon cancer cells (Kim et al, 2008
). A study using melanoma cells (Zheng et al, 2009
) has shown that the MAPK3/1 pathway phosphorylates STK11 on Ser325 and Ser428 and promotes the uncoupling of AMPK from STK11, which negatively regulates AMPK. Regulation of AMPK activity by the MAPK3/1 pathway, independent of STK11 Ser428 phosphorylation, has also been reported (Esteve-Puig et al, 2009
). In fibroblast cells, AMPK differentially inhibits the MAPK3/1 pathway by inhibiting RAS activation or stimulating the RAS-independent pathway in response to cellular energy status (Kim et al, 2001
). We should also consider the complex TSC2-MTOR axis-mediated linkage. AMP-activated protein kinase suppresses MTOR activity directly by phosphorylating MTOR at Thr2446 and indirectly by phosphorylating TSC2 at Thr1227 and Ser1345 and increasing the activity of TSC-complex (Inoki and Guan, 2009
). MAPK3/1 increases MTOR activity by phosphorylating TSC2 at Ser540 and Ser664, which causes the attenuation of TSC2 (Ma et al, 2005
). Our findings may support the hypothesis that AMPK activation can make a strong impact on tumour behaviour as the ‘brake' only when MAPK3/1 is active. Additional studies are needed to confirm our findings and elucidate the exact mechanism of effect of MAPK3/1 on AMPK to modify tumour behaviour.
Our study has shown that MAPK3/1 activation has a differential effect on patient mortality according to AMPK status; p-MAPK3/1 expression is associated with good prognosis among p-AMPK-positive patients, but with poor prognosis among p-AMPK-negative patients. It remains controversial how MAPK3/1 activation affects behaviour of different cancers (Milde-Langosch et al, 2005
; Pelloski et al, 2006
). A study on 135 colorectal cancers has shown that p-MAPK3/1 expression is associated with poor prognosis (Schmitz et al, 2007
). In contrast to that study (N
=135), our study evaluated the expression status of both p-MAPK3/1 and p-AMPK in a much larger cohort of 718 colorectal cancers. In addition, we assessed the interactive effect of p-MAPK3/1 and p-AMPK expression independent of other molecular events that have been documented to be critical in colorectal carcinogenesis.
Recently, AMPK has been proposed as a potential target for cancer prevention and treatment, and various AMPK activators have been preclinically assessed (Fay et al, 2009
). Among them, metformin, a widely used anti-diabetic drug, has shown promising results (Buzzai et al, 2007
; Zakikhani et al, 2008
). Metformin may have two properties of potential oncologic relevance: it has a direct, STK11-AMPK pathway-dependent growth inhibitory effect and decreases systemic insulin levels (Pollak, 2008
). Interestingly, two observational studies have shown that diabetic patients treated with metformin experienced a lower incidence of any kind of cancer and a lower cancer-related mortality (Evans et al, 2005
; Bowker et al, 2006
). Hereafter, in clinical trial of this drug, examining AMPK status in cancer tissue might be important. In this regard, our findings may have clinical implications. In addition, drugs targeting the MAPK3/1 pathway are intensively being developed and tested in clinical trials for various human cancers (Beeram et al, 2005
). Although the usefulness of MAPK3/1 expression as a biomarker for sensitivity to these drugs is uncertain (Yeh et al, 2009
), further understanding of the linkage between the AMPK and MAPK3/1 pathways could potentially provide useful information for refinement of therapeutic strategies.
We found significant relations of p-AMPK expression with MSI-high and CIMP-high. MSI and CIMP status reflect global genomic and epigenomic aberrations in tumour cells, and hence, are associated with various clinical, pathologic and molecular features (Ogino and Goel, 2008
). Considering the known relationship between MSI and/or CIMP and molecular alterations related to energy metabolism (Ogino et al, 2007b
; Nosho et al, 2009
), MSI and CIMP may influence energy sensing status of cancer cells.
There are limitations in this study. For example, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use substantially differed according to AMPK status in tumour, because such data were unavailable for treatment decision making. In addition, our multivariate survival analysis finely adjusted for disease stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV, unknown), on which treatment decision making was mostly based. As another limitation, beyond cause of mortality, data on cancer recurrence were unavailable in these cohort studies. Nonetheless, colorectal cancer-specific survival might be a reasonable surrogate of colorectal cancer-specific outcome. Furthermore, the cutoffs for p-AMPK and p-MAPK3/1 used in this current study need to be validated in an independent data set.
There are advantages in utilising the database of the two prospective cohort studies, the Nurses' Health Study and the Health Professionals Follow-Up Study, to examine prognostic significance of tumour AMPK expression. Anthropometric measurements, family history, cancer staging, and other clinical, pathologic, and tumour molecular data were prospectively collected, blinded to patient outcome. Cohort participants who developed cancer were treated at hospitals throughout the United States, and thus more representative colorectal cancers in the US population than patients in one to several academic hospitals. There was no demographic difference between cases with tumour tissue analysed and those without tumour tissue analysed (Chan et al, 2007
). Finally, our rich tumour database enabled us to simultaneously assess pathologic and tumoral molecular correlates and control for confounding by a number of tumoral molecular alterations.
In summary, we have shown that AMPK activation is associated with good prognosis among MAPK3/1-activated colorectal cancer patients, while AMPK activation is not associated with prognosis among MAPK3/1-inactive cancer patients. Additional studies are necessary to confirm our observations and to elucidate exact mechanisms by which AMPK and MAPK3/1 interact and affect tumour behaviour. This possible interaction between the AMPK and MAPK3/1 pathways may have considerable implications because both pathways are potential targets for cancer treatment and prevention. In this regard, examining AMPK and MAPK3/1 status in cancer tissue may be important in future clinical trials.