Circulating adiponectin is inversely associated with colorectal carcinoma. However, adiponectin receptor expression has not been examined in normal gastrointestinal tissue, colorectal malignancies, or gastrointestinal stromal tumors (GISTs). We collected 40 colorectal carcinomas and 12 non-tumor colorectal tissue specimens from patients with colorectal cancer, as well as 45 tumor and 13 non-tumor specimens from patients with GIST. Expression and localization of adiponectin receptors (AdipoR1 and AdipoR2) were assessed using immunohistochemistry. We also confirmed expression of adiponectin receptors using rtPCR in matched normal and colorectal cancer specimens obtained from five patients. Finally, we detected adiponectin receptors and assessed adiponectin signaling in three colon cancer cell lines. Adiponectin receptor expression, assessed by either rtPCR or immunohistochemistry, was present in normal tissue and was significantly lower than in colorectal carcinomas. Among carcinomas, 95% displayed positive or strongly positive expression of AdipoR1 and 88% of AdipoR2, versus 8% and 0%, respectively, for non-tumor specimens (P!0.0001). AdipoR1 expression assessed by rtPCR was 1.6-fold higher in tumor than in non-tumor tissue (P!0.05). In addition, we found that adiponectin at physiological concentrations can activate in vitro intracellular signaling pathways in three colon cancer cell lines,

Purpose: The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis.Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. Experimental Design: We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens.Results: All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor nB (NF-nB) inhibitor InB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-nB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain^like interleukin-1h^converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal^regulated kinase kinase/extracellular signal^regulated kinase, NF-nB, and activator protein-1 pathways in thyroid carcinoma cells in vitro ; and protected cells from tumor necrosis factor^related apoptosis-inducing ligand^induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. Conclusions: In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

Thyroid carcinoma cells overall are poorly responsive to clinically relevant concentrations of AEE788 in vitro. The presence of EGFR-activating TK domain mutations may identify a small minority of thyroid cancer patients that may benefit from EGFR inhibitors, but additional preclinical evidence of efficacy is needed.

B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAF V600E specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAF V600E patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-Raf V600E induced a comparable reduction of viability in both wild-type and BRAF V600E mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAF V600E -harboring cells than wild-type ones, possibly because of better inhibitory activity against B-Raf V600E . We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-Raf V600E may provide clinical benefit for patients with thyroid cancer.

The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK pathway that relays extracellular signals for cell proliferation and survival. Several types of human malignancies harbor activating BRAF mutations, most frequently a V600E substitution. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small cell lung carcinomas harboring activating EGFR TK domain somatic mutations. We evaluated the presence of mutations in BRAF (exons 11 and 15), KRAS (exons 1 and 2), NRAS (exons 1 and 2), and EGFR (exons 18-21) in adrenal carcinomas (35 tumor specimens and two cell lines) by DNA sequencing. BRAF mutations were found in two carcinomas (5.7%). Four carcinomas (11.4%) carried EGFR TK domain mutations. One specimen carried a KRAS mutation, and another carried two NRAS mutations. No mutations were found in the two adrenocortical cell lines. BRAF-and EGFR-mutant tumor specimens exhibited stronger immunostaining for the phosphorylated forms of the MEK and ERK kinases than their wild-type counterparts. EGFR-mutant carcinomas exhibited increased phosphorylation of EGFR (Tyr 992) compared with wild-type carcinomas. We conclude that BRAF, RAS, and EGFR mutations occur in a subset of human adrenocortical carcinomas. Inhibitors of the Ras/Raf/MEK/ERK and EGFR pathways represent candidate targeted therapies for future clinical trials in carefully selected patients with adrenocortical carcinomas harboring respective activating mutations.