Blockade of Fatty Acid Synthase Triggers Significant Apoptosis in Mantle Cell Lymphoma

Fatty acid synthase (FASN), a key player in the de novo synthetic pathway of long-chain fatty acids, has been shown to contribute to the tumorigenesis in various types of solid tumors. We here report that FASN is highly and consistently expressed in mantle cell lymphoma (MCL), an aggressive form of B-cell lymphoid malignancy. Specifically, the expression of FASN was detectable in all four MCL cell lines and 15 tumors examined. In contrast, benign lymphoid tissues and peripheral blood mononuclear cells from normal donors were negative. Treatment of MCL cell lines with orlistat, a FASN inhibitor, resulted in significant apoptosis. Knockdown of FASN expression using siRNA, which also significantly decreased the growth of MCL cells, led to a dramatic decrease in the cyclin D1 level. β-catenin, which has been previously reported to be upregulated in a subset of MCL tumors, contributed to the high level of FASN in MCL cells, Interesting, siRNA knock-down of FASN in turn down-regulated β-catenin. In conclusion, our data supports the concept that FASN contributes to the pathogenesis of MCL, by collaborating with β-catenin. In view of its high and consistent expression in MCL, FASN inhibitors may hold promises for treating MCL

Interleukin 22 Signaling Promotes Cell Growth in Mantle Cell Lymphoma

AbstractMantle cell lymphoma (MCL) is a specific type of aggressive B-cell non-Hodgkin lymphoma. We recently found that IL-22RA1, one of the two subunits of the interleukin 22 (IL-22) receptor, is expressed in MCL cell lines but not benign lymphocytes. In view of normal functions of IL-22 signaling, we hypothesized that the aberrant expression of IL-22RA1 may contribute to the deregulation of various cell signaling pathways, thereby promoting cell growth in MCL. In this study, we first demonstrated the expression of IL-22RA1 in all three MCL cell lines and eight frozen tumors examined using reverse transcription-polymerase chain reaction and Western blot analysis. In support of the concept that IL-22 signaling is biologically important in MCL, we found that MCL cells treated with recombinant IL-22 had a significant increase in cell growth that was associated with STAT3 activation. To investigate the mechanism underlying the aberrant expression of IL-22RA1, we analyzed the gene promoter of IL-22RA1, and we found multiple binding sites for NF-κB, a transcriptional factor strongly implicated in the pathogenesis of MCL. Pharmacologic inhibition of NF-κB resulted in a substantial reduction in the level of IL-22RA1 protein expression in MCL cells. To conclude, IL-22RA is aberrantly expressed in MCL, and we have provided evidence that IL-22 signaling contributes to the pathogenesis of MCL

FASN was highly expressed in MCL cell lines.

<p>A) RT-PCR studies demonstrated the high level of <i>FASN</i> mRNA in four MCL cell lines. SKBR3 (a breast cancer cell line) and HepG2 (a hepatocarcinoma cancer cell line) were used as positive controls. B) The FASN protein expressed in MCL cell lines was readily detectable by Western blots. SKBR3 was used as a positive control. In contrast, protein lysates prepared from isolated peripheral blood mononuclear cells (PBMCs) from a healthy donor were negative for the FASN protein. C) Flow cytometry analysis revealed the expression of FASN in Jeko-1 cells.</p

Knockdown of FASN decreased the expression of cyclin D1 and induced cell death of MCL cells.

<p>A) Two MCL cell lines, Mino and SP53, were treated with siRNA to knockdown FASN. The expression of FASN and cyclin D1 were assessed by western blot. The expression of FASN and cyclin D1 was dramatically decreased after <i>FASN</i> siRNA treatment. B) Quantification studies showed that FASN expression was decreased in MCL after treatment with <i>FASN</i> siRNA as compared to cells treated with scramble siRNA. Triplicate experiments were performed. (C) Two MCL cell lines were treated with <i>FASN</i> siRNA for up to 96 hours. The cell growth was evaluated using trypan blue exclusion assay. Knockdown of FASN expression significantly decreased the growth of MCL cells (for Mino cells - ^# p<0.005 at 72 hours; ^## p<0.005 at 96 hours; for SP53 cells - * p<0.05 at 72 hours; ** p<0.05 at 96 hours). D) Treatment of two MCL cell lines with <i>FASN</i> siRNA significantly decreased the number of viable cells, as assessed by using the MTS assay. The differences are statistically significant for Mino (^# p<0.05 at 48 hours and ^## p<0.005 at 72 hours) and for SP53 (* p<0.05 at 48 hours and ** p<0.005 at 72 hours). Triplicate experiments were performed. E) Treatment of SP53 cell line with siRNA <i>FASN</i> induced caspase 3 cleavage.</p

β-catenin decreased USP2a protein expression in MCL cells.

<p>A and B) two MCL cell lines were treated with two different siRNA β-catenin species or scramble siRNA, and the expression of phospho-mTor (ser2481) and USP2a were evaluated by Western blots. siRNA treatment induced a substantial decrease in the levels of the USP2a protein in both cell lines. In contrast, no appreciable modulation of phospho-mTor (as a surrogate marker of mTor activation) was observed. Triplicate experiments were performed and represented results are illustrated.</p

FASN inhibitors induced apoptosis of MCL cells.

<p>The four MCL cell lines were treated with two different concentrations of Orlistat. A) The degree of apoptosis was assessed based on the cell surface expression of Annexin V in MCL cell lines detectable by flow cytometry. In addition, the cell viability in two MCL cells, Rec-1(B) and Mino (C) treated with 5 µM or 10 µM Orlistat for 48 hours, was significantly correlated with the enzymatic activity of caspase 3/7. D) Using western blot, cleaved capase 3 and PARP were detectable in MCL cells treated with 10 µM Orlistat.</p

Orlistat induced cell death in primary leukemic MC cells.

<p>A) Leukemic cells from three patients were treated with two different concentrations of Orlistat. The number of viable cells was assessed by trypan blue assay after 24 hours of treatment. Results illustrated represented the ‘pooled’ data of these three samples. B) In the same experiment, the caspase-3/7 activity was assessed using a commercially available Caspase-3/7 Apo-One kit. Again, results illustrated represented the ‘pooled’ results of three samples. Triplicate experiments were performed. C) Representative results of propidium iodine staining of leukemic MCL samples treated with Orlistat showed a dose-dependent increase of staining detectable by flow cytometry.</p

FASN inhibition decreased β-catenin expression.

<p>The MCL cell line Mino was treated with Orlistat (A) or siRNA (B) to inhibit or knock-down FASN, respectively. The β-catenin protein expression was determined by western blot after 48 hours of treatment. FASN inhibition by either Orlistat or siRNA substantially decreased the protein expression of β-catenin. Triplicate experiments were performed.</p