BACKGROUND-The PI3K/Akt/mTOR pathway plays a critical role in the growth and progression of colorectal cancer (CRC). The purpose of our study was twofold: 1) to determine the expression levels of several key components of this pathway including p85α, Akt1, Akt2, pmTOR Ser2448 and p-p70S6K Thr389 in CRCs, and 2) to correlate the expression of these proteins with cancer stage and location (left-vs. right-sided).

Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Understanding the distinct genetic and epigenetic changes contributing to the establishment and growth of metastatic lesions is crucial for the development of novel therapeutic strategies. In a search for key regulators of colorectal cancer metastasis establishment, we have found that the serine/ threonine kinase Akt2, a known proto-oncogene, is highly expressed in late-stage colorectal cancer and metastatic tumors. Suppression of Akt2 expression in highly metastatic colorectal carcinoma cells inhibits their ability to metastasize in an experimental liver metastasis model. Overexpression of wild-type Akt1 did not restore metastatic potential in cells with downregulated Akt2, thus suggesting non-redundant roles for the individual Akt isoforms. In contrast, Akt2 overexpression in wild-type PTEN expressing SW480 colorectal cancer cells led to the formation of micrometastases; however, loss of PTEN is required for sustained formation of overt metastasis. Finally, we found that the consequence of PTEN loss and Akt2 overexpression function synergistically to promote metastasis. These results support a role for Akt2 overexpression in metastatic colorectal cancer and establish a mechanistic link between Akt2 overexpression and PTEN mutation in metastatic tumor establishment and growth. Taken together, these data suggest that Akt family members have distinct functional roles in tumor progression and that selective targeting of the PI3K/Akt2 pathway may provide a novel treatment strategy for colorectal cancer metastasis.AKT2 ͉ colorectal cancer ͉ metastasis ͉ oncogene ͉ therapeutic target

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Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.Tumor suppressor protein p53 keeps us free of cancer when it is functional. Mice lacking p53 (p53 Ϫ/Ϫ ) spontaneously develop numerous neoplasms within 6 months (1). Mutated p53 genes are seen in more than 50% of all human cancers, making them the most frequently observed genetic derangement in human cancer (2). At a molecular level, the ability of p53 to eliminate cancerous cells relies on its ability to induce apoptosis, through either the transcriptional activation of proapoptotic genes such as Noxa (3), PUMA 4 (4), and Bax (5) or the direct transcription-independent activation of Bax on mitochondria (6). Growing cancers manage to keep p53 in check either by mutating the p53 gene itself (7-9) or by expressing p53 inhibitors such as Mdm2 (9, 10). The function of fortilin, a ubiquitous, highly conserved, 172-amino acid polypeptide also known as "translationally controlled tumor protein," or TCTP, remained unknown (11,12). Investigation in our laboratory and others showed that fortilin possesses potent anti-apoptotic activity (13-15). Fortilin is overexpressed in human cancers (16,17), the depletion of which is associated with spontaneous death of cancerous cells (13, 18). Higher levels of fortilin are associated with more malignant cancer phenotypes (14). Although heterozygous fortilin-deficient mice (fortilin ϩ/Ϫ ) were normal in appearance and fertile, homozygous fortilin-deficient (fortilin Ϫ/Ϫ ) mice were embryonically lethal around 3.5 days postcoitus due to massive apoptosis, as reported by our laboratory and others (19 -21).The mechanism by which fortilin functions as an anti-apoptotic molecule has been under robust investigation. First, based on the fact that fortilin physically interacts with myeloid cell leukemia protein-1 (MCL1), an anti-apoptotic Bcl-2 family member, it was suggested that fortilin stabilizes and exerts its anti-apoptotic activity through MCL1 (22). However, fortilin is capable of protecting cells from apoptosis in the...

Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.

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