We conducted this study to examine the relationship between phosphorylated AKT (p-AKT) expression and patient survival in a large cohort of stage I-IV colorectal cancers. AKT has emerged as a central node in cell signaling pathways, downstream of growth factors, cytokines, and other cellular stimuli.1-3
AKT is aberrantly activated in a wide variety of human cancers and is increasingly important as a promising target for cancer therapy.4-6
We have found that p-AKT expression in colorectal cancer is associated with early stage and favorable prognosis, suggesting that p-AKT may be a biomarker to identify patients with superior outcome and a possible therapeutic target (analogous to estrogen receptor in breast cancer).
Examining tumor factors and clinical outcome is important in cancer research.35-39
Studies examining the relation between p-AKT expression and prognosis in human cancers have yielded variable results.7-12
Previous studies on colorectal cancer have suggested no prognostic role of p-AKT expression.15-18
Notably, in the study with over 1000 colorectal cancers,15
tumoral p-AKT expression was associated with early nodal stage, but not independently with patient survival. However, that study15
mutation data. A difference in the prognostic data by that study15
and our current study might be due to a difference in the patient cohorts or the methods to assess p-AKT expression, or simply due to a chance variation between independent studies. In addition, all but one15
were limited by small sample sizes (N<160). Our current study (N=717) has shown that p-AKT expression is associated with PIK3CA
mutation as expected from their causal link, and that our method and cutoff for p-AKT positivity are reasonable for evaluating AKT activation levels in paraffin-embedded tissues. Furthermore, in contrast to the prior studies,15-18
we assessed the prognostic effect of p-AKT expression independent of PIK3CA
mutation and other molecular events that have been documented to be critical in colorectal carcinogenesis.
Considering experimental data suggesting that AKT plays critical roles in tumor proliferation, survival, invasion, and angiogenesis,1-3, 40
one would expect that p-AKT expression would imply poor prognosis. It is very common to conceive that the presence of oncogene activation (or tumor suppressor inactivation) should imply aggressive tumor behavior. However, this preconception does not always hold true. Colon cancers develop through accumulation of multiple genetic and epigenetic events, and each tumor has its own unique combination of molecular aberrations. Some tumors activate AKT, while others do not. In order to acquire malignant characteristics, those AKT-inactive tumors need to have aberrations which can be an alternative to AKT activation; those aberrations may lead to more aggressive behavior than AKT activation actually does. This is well exemplified by the association between estrogen receptor (ESR1, or ER-alpha) expression in breast cancer and good prognosis. ESR1 is known to contribute to breast cancer development; yet ESR1 expression marks tumors with favorable outcome.41
This is probably because breast cancer without ESR1 expression might have developed through more detrimental events than ESR1 expression. Another example is MSI in colorectal cancer. MSI is known to cause inactivation of a number of tumor suppressors; yet MSI marks tumors with good prognosis.42
The aforementioned preconception that oncogene activation (or tumor suppressor inactivation) should be associated with poor outcome can cause serious publication bias, because reports consistent with this preconception have been regarded favorably during journal's decision process, whereas reports inconsistent with the preconception have been treated unfavorably.43
Another possible explanation for the relationship between AKT activation and good prognosis may be due to “tumor suppressive” roles of AKT.44
AKT has blocked cancer cell mortality and invasion through the transcription factor NFAT 45
or down-regulation of RHO activity.46
Another study has reported that AKT1 activation can promote tumorigenesis but suppresses tumor invasion.47
The inhibitory effect of AKT activation on cancer cell cycle has been reported.48
In addition, it is possible that each AKT isoform may have different functions.49, 50
Future studies are necessary to confirm our observations as well as to elucidate biological mechanisms by which AKT activation affects colorectal tumor behavior.
Interestingly, we found that p-AKT expression was independently associated with FASN expression and low tumor grade; the latter association is consistent with a previous study.15
Inhibition of FASN has resulted in the down-regulation of AKT pathway.20
The PI3K/AKT pathway activation has modulated the expression and/or nuclear maturation of the transcription factor SREBF1, stimulating FASN expression.20
Our finding of the relationship between p-AKT and FASN expression may be consistent with these experimental data.
There are limitations in this study. For example, data on cancer treatment were unavailable. Nonetheless, it is unlikely that chemotherapy use substantially differed according to AKT status in tumor, since such data were unavailable for treatment decision making. In addition, our multivariate survival analysis adjusted for disease stage as finely as possible (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 recurrences were unavailable in these cohort studies. Nonetheless, colorectal cancer-specific survival might be a reasonable surrogate of colorectal cancer-specific outcome.
Immunohistochemical evaluation of p-AKT expression in cancer tissue has been a challenge, and there is no standardized method. Pre-analytical variables such as tissue processing may have considerable impact on antigenicity of p-AKT, which may be substantially influenced by a slight difference in conditions of immunohistochemical procedure. We tried to minimize such an external source of noise in a number of ways. We constructed tissue microarray (TMA) and performed the immunohistochemical procedure in a very similar condition for all specimens. The quality of tissue sections, which depends on age of tissue and time after cutting the TMA blocks, should be considered. In our current study, p-AKT expression was not associated with age of tissue (i.e., year of diagnosis), supporting that p-AKT antigen might not substantially be deteriorated over time. With regard to time after block cutting, as we cut all TMA blocks into sections around the same time, time after block cutting was uniform from case to case. In addition, we obtained four tissue cores from each case to overcome within-tumor heterogeneity. Interpretation of protein expression took into account background noise if any, and we calibrated against such background noise in each case. Furthermore, any pre-analytical variability was largely non-differential (i.e., random) in nature, and thus, might have conservatively biased our results towards the null hypothesis. Because of a large sample size, we were still able to detect the biologically-reasonable relationship between PIK3CA mutation and p-AKT expression as well as the relations of p-AKT expression with disease stage and prognosis. The cutoff for p-AKT used in this current study needs to be validated in an independent dataset.
There are advantages in utilizing the database of the two prospective cohort studies, the Nurses' Health Study and the Health Professionals Follow-up Study, to examine prognostic significance of tumor biomarkers. Anthropometric measurements, family history, cancer staging, and other clinical, pathologic, and tumoral molecular data were prospectively collected, blinded to patient outcome. Cohort participants who developed cancer were treated at hospitals throughout the U.S. (in 48 States except for North Dakota and Alaska), and thus more representative colorectal cancers in the general U.S. population than patients in one to a few academic hospitals. There were no demographic difference between cases with tumor tissue analyzed and those without tumor tissue analyzed 22
. Finally, our rich tumor database enabled us to simultaneously assess pathologic and tumoral molecular correlates and control for confounding by a number of tumoral molecular alterations.
In summary, our large cohort study has shown that p-AKT expression in colorectal cancer is associated with PIK3CA mutation, early disease stage and favorable prognosis. The high percentage of p-AKT positive patients in this study certainly supports a vital role of AKT activation in the pathogenesis of colorectal cancer. Our findings suggest a possibility that AKT activation may have a predictive role in a manner analogous to estrogen receptor (ESR1) in breast cancer, identifying a subgroup of patients with better prognosis and serving as a therapeutic target at the same time.