AKT activation up-regulates insulin-like growth factor I receptor expression and promotes invasiveness of human pancreatic cancer cells.

Insulin-like growth factor I receptor (IGF-IR) is frequently overexpressed in several types of human malignancy and is associated with invasion and metastasis of tumor cells. Recently, IGF-IR expression was reported to be up-regulated in the human pancreatic cancer cell line PANC-1 when cells were stably transfected with active Src. The downstream targets of Src that lead to the up-regulation of IGF-IR expression were previously unknown. We demonstrate here that AKT regulates IGF-IR expression in PANC-1 and AsPC-1 cells. Cells transfected with active Src exhibited significantly more IGF-IR protein compared with vector-transfected cells. Overexpression of wild-type or constitutively active AKT (i.e., AKT1 or AKT2) also resulted in elevated IGF-IR expression. IGF-IR protein levels were higher in cells transfected with constitutively active AKT than in cells transfected with active Src. In vitro kinase assays showed that AKT kinases are activated by active Src and inhibited by dominant negative Src or the tumor suppressor PTEN. Furthermore, AKT-induced IGF-IR expression was down-regulated by dominant-negative Src or PTEN. In addition, cells transfected with activated AKT in the presence of IGF-I were shown to have enhanced invasiveness compared with control cells. These data provide evidence for a link between AKT signaling and the regulation of IGF-IR expression and demonstrate that active AKT promotes the invasiveness of pancreatic cancer cells through the up-regulation of IGF-IR expression.

p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma.

To determine whether p16 is altered in human malignant mesothelioma (MM), molecular analysis of multiple 9p loci was performed on 40 cell lines and 23 primary tumors from 42 MM patients. We identified homozygous deletions of p16 in 34 (85%) cell lines and a point mutation in 1 line. Down-regulation of p16 was observed in 4 of the remaining cell lines, 1 of which displayed a DNA rearrangement of p16. Homozygous deletions of p16 were identified in 5 of 23 (22%) primary tumors; no mutations or rearrangements were found in these specimens. Four cell lines displayed a single homozygous deletion proximal to or distal to p16; 4 others had 2 nonoverlapping deletions, one involving p16 and the other involving a region proximal to this locus. These data indicate that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors. Furthermore, deletions of 9p21-p22 outside of the p16 locus may reflect the involvement of other putative tumor suppressor genes that could also contribute to the pathogenesis of some MMs.

Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene.

We isolated mouse cDNA clones containing the entire coding region of the putative oncogene Akt2. Sequence analysis revealed that, like its human homolog, Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain at its amino terminus. Fluorescence in situ hybridization of the mouse cDNA to rodent metaphase spreads demonstrated that the Akt2 gene maps to mouse chromosome band 7B1 and rat chromosome 1q22. Expression levels of mouse Akt2 mRNA and Akt2 protein varied among tissues, with the highest levels in skeletal muscle. Akt2 expression was low in a multipotent fibroblast cell line, but it was upregulated when these cells were transformed with Myod and induced to differentiate into myocytes. These data demonstrate that Akt2 expression is activated during cellular differentiation and suggest that it functions in the signaling pathways of some adult tissues.

Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin.

Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling molecules. Although there has been extensive interest in the mechanism by which the closely-related Akt kinase participates in phosphatidylinositol 3-kinase-mediated signaling, comparatively little is known regarding the expression and function of Akt2. This manuscript is the first to describe Akt2 mRNA expression in the developing mouse and the activation of AKT2 by insulin. These studies demonstrate that Akt2 is especially abundant in brown fat and, to a lesser extent, skeletal muscle and liver, tissues which are highly insulin-responsive and play a role in glucose metabolism. Endogenous Akt2 expression also is upregulated in fully-differentiated C2C12 myotubes and 3T3-L1 adipocytes, suggesting that these murine cell lines represent useful in vitro models for studies of Akt2 function. We show that HA-tagged AKT2 is activated in response to insulin stimulation in vitro and that activation of AKT2 is not induced in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These data suggest that Akt2 expression is fundamental to the differentiated state of fat and muscle cells and that activation of AKT2 kinase by insulin is mediated through the phosphatidylinositol 3-kinase signaling pathway.

Transforming activity and mitosis-related expression of the AKT2 oncogene: evidence suggesting a link between cell cycle regulation and oncogenesis.

The AKT2 oncogene encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling proteins. Recently, it was shown that AKT2 kinase activity can be induced by platelet-derived growth factor through phosphatidylinositol-3-OH kinase, suggesting that AKT2 may be an important signal mediator that contributes to the control of cell proliferation. We previously reported amplification and overexpression of AKT2 in human cancers. To investigate the transforming activity of AKT2, we used a retrovirus-based construct to express AKT2 in NIH3T3 cells. Overexpression of AKT2 was found to transform NIH3T3 cells, as determined by growth in soft agar and tumor formation in nude mice. The oncogenic activity of AKT2 was diminished by truncation of a 70-amino acid proline-rich region at the carboxyl-terminus. To facilitate the characterization of AKT2, we generated monoclonal and polyclonal antibodies against this protein. AKT2 was localized to the cytoplasm by cell fractionation experiments, immunocytochemistry, and immunofluorescence. Protein levels were more abundant in mitotic cells than in interphase cells. Western blot analysis of synchronized pancreatic cancer cells demonstrated that the expression level of AKT2 protein in mitotic cells is three to fivefold higher than in their interphase counterparts. A time-course study of phytohemagglutinin-stimulated lymphocytes revealed that AKT2 mRNA and AKT2 protein levels are highest 48-72 h after addition of mitogen, when cells are actively dividing. These data suggest that AKT2 could play a significant role in cell cycle progression and that the oncogenic activity of overexpressed AKT2 may be mediated by aberrant regulation of cellular proliferation.

Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling.

The AKT family of serine threonine kinases is of critical importance with regard to growth factor signaling, cell proliferation, survival and oncogenesis. Engagement of signaling receptors induces the lipid kinase, phosphatidylinositol 3-kinase (PI3K), which enables the activation of AKT. Responsive to the PI3K/AKT pathway is the mammalian target of rapamycin (mTOR), a major effector that is specifically implicated in the regulation of cell growth as a result of nutrient availability and cellular bioenergetics. These kinases mediate the activity of a multitude of intracellular signaling molecules and intersect with multiple pathways that regulate cellular processes. Elucidating the role of AKT/mTOR in metabolism and in hallmark signaling pathways that are aberrantly affected in cancer has provided a solid foundation of discoveries. From this, new research directions are emerging with regard to the role of AKT/mTOR in diabetes and T cell-mediated immunity. As a result, a new perspective is developing in how AKT/mTOR functions within intracellular signaling pathways to maintain cellular homeostasis. An appreciation is emerging that altered equilibrium of AKT/mTOR pathways contributes to disease and malignancy. Such new insights may lead to novel intervention strategies that may be useful to reprogram or reset the balance of intracellular signaling.

Molecular cloning and localization of the human GAX gene to 7p21.

The GAX homeobox gene is expressed in the cardiovascular tissues of the adult rat, including heart, lung, kidney, and blood vessels. In the vasculature it is specifically expressed in quiescent smooth muscle cells, but its expression is rapidly down-regulated when these cells are stimulated to proliferate with mitogens. Since vascular smooth muscle cell proliferation is important in the pathology of blood vessel disorders, the human GAX gene was isolated and characterized. The human GAX cDNA was obtained by an anchored-PCR approach using cDNA templates from cardiovascular tissues and amplification primers designed from sequence information of the rat GAX cDNA and the homeodomain-containing exon of the human GAX gene. The human and rat GAX gene coding sequences are 98% conserved at the amino acid level and 83% conserved at the nucleotide level. Similar to rat, the human homolog contains a CAX trinucleotide repeat N-terminal to the homeodomain that encodes for a stretch of 17 consecutive histidine or glutamine residues. The human GAX locus was mapped by fluorescence in situ hybridization to the short arm of chromosome 7 at band p21. The human cDNA sequence will be useful for analyses of GAX gene expression in cardiovascular diseases.

Chromosome mapping of the mouse Akt2 gene and Akt2 pseudogene.

We previously reported the cloning of a murine cDNA encoding the protein-serine/threonine kinase Akt2, and we used this clone to map the Akt2 gene to mouse chromosome (MMU) 7B1 by fluorescence in situ hybridization. We now have cloned and partially sequenced a mouse Akt2 pseudogene. An analysis of two sets of multilocus crosses revealed that the Akt2 gene is closely linked to the Cyp2a locus in proximal MMU7. The Akt2 pseudogene was mapped to proximal MMU11 by both multilocus mapping and fluorescence in situ hybridization.

Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.

A common region of deletion on chromosome 17q in both sporadic and familial epithelial ovarian tumors distal to BRCA1.

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17p13.1 and 17p13.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17q12-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans approximately 25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.

Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas.

The AKT2 gene is one of the human homologues of v-akt, the transduced oncogene of the AKT8 virus, which induces lymphomas in mice. In previous studies, AKT2, which codes for a serine-threonine protein kinase, was shown to be amplified and overexpressed in some human ovarian carcinoma cell lines and amplified in primary tumors of the ovary. To confirm and extend these findings, we conducted a large-scale, multicenter study of AKT2 alterations in ovarian and breast cancer. Southern-blot analysis demonstrated AKT2 amplification in 16 of 132 (12.1%) ovarian carcinomas and in 3 of 106 (2.8%) breast carcinomas. No AKT2 alteration was detected in 24 benign or borderline tumors. Northern-blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas which were negative for AKT2 amplification. The difference in the incidence of AKT2 alterations in ovarian and breast cancer suggests a specific role for this gene in ovarian oncogenesis. No significant association was found between AKT2 amplification and amplification of the proto-oncogenes MYC and ERBB2, suggesting that amplification of AKT2 defines an independent subset of breast and ovarian cancers. Ovarian cancer patients with AKT2 alterations appear to have a poor prognosis. Amplification of AKT2 was especially frequent in undifferentiated tumors (4 of 8, p = 0.019), suggesting that AKT2 alterations may be associated with tumor aggressiveness.