The Role Of BIN1 Tumor Suppressor Isoforms In Regulation Of Proliferation, Apoptosis and Tumor Formation Of Human Cutanous T-Cell Lymphoma Cells In Vitro and In Vivo

Cutaneous T-cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders that are characterized by homing of malignant T-cells to the surface of skin. There are two main types of CTCL: Mycosis Fungoides (MF) and its leukemic variant Sezary Syndrome (SS), which together represent about 65-70% of all CTCL cases. The precise genetic pathogenesis of these diseases remains largely undetermined. Recently, our research group has demonstrated that AHI-1 (Abelson Helper Integration site-1) oncogene is involved in CTCL. Expression of AHI-1 is increased in human leukemia cell lines, with marked upregulation (up to 40 fold) in CTCL lines (Hut78 and Hut102). Moreover, in FACS-purified CD4⁺CD7^- Sezary cells from patients with Sezary Syndrome, AHI-1 expression is higher at both the RNA and protein levels compared to normal CD4⁺ cells. Furthermore, stable suppression of endogenous AHI-1 in Hut78 cells using small interfering RNA, normalizes their transforming activity both in vitro and in vivo. Thus, lymphomagenic activity of Hut78 cells is partially dependent on the expression of AHI-1. Interestingly, BIN1 (Bridging integrator 1) was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, whereas the BIN1 (+12A) behaves as a cancer-related isoform in solid tumor models. However, the role of BIN1 in regulation of normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4⁺CD7^- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into two CTCL cell lines, Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell assays and ³H-Thymidine uptake assays (2-3 fold, p<0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 fold, p<0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. These findings show anti-proliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells. In addition, subcellular fractionation and confocal microscopy further indicated that both the BIN1 (+10, +13) and BIN1 (+12A) isoforms are mainly located in the nucleus in Hut78 and HH cells. Furthermore, to investigate the effects of overexpression of BIN1 isoforms on the ability to induce tumors in vivo, we tested their leukemogenic potential by injecting transduced HH cells into non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice. Mice injected subcutaneously with either parental HH or control empty vector cells (2 x 10⁷/per mouse), showed local tumor formation in 6 of 6 mice within 4 days post-injection. The local tumors enlarged progressively and were often 1.5–2 cm in diameter by 3 weeks after injection. In contrast, no local tumors formed in mice given injections of equal numbers of BIN1-transduced HH cells after 14 days, in 12 out of 12 mice. Tumor formation was only observed in BIN1-transduced HH cells after 3 weeks post-injection. However, the local tumors were significantly smaller in BIN1-transduced HH cells compared to controls (∼4-fold). These findings further indicate that the two BIN1 isoforms have tumor suppressor activities in NOD/SCID mice and can significantly delay tumor formation and reduce tumor size in vivo.