Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across anatomic barriers and colonize distant organs. Here we show an association between metastasis and expression of a type I receptor-tyrosine-kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal post-partum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high-levels ROR1 were more likely to have gene-expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low-levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast-cancer cell-lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g. vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g. CK-19), and tight-junction proteins (e.g. ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immune-deficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a mAb specific for ROR1 induced down-modulation of vimentin, and inhibited cancer-cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis, and that antibodies targeting ROR1 can inhibit cancer progression and metastasis.
Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21 WAF1/Cip1 (p21).Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL. (Blood. 2014;124(10):1637-1644 IntroductionLenalidomide is a second-generation immunomodulatory drug (IMiD) 1-3 that has both direct tumoricidal, as well as immunomodulatory activity in patients with multiple myeloma. 4 This drug also has clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not directly cytotoxic to CLL cells in vitro. 5,6 As such, its clinical activity in CLL is presumed to be secondary to its immune modulatory activity. 7 Indeed, lenalidomide indirectly modulates CLL-cell survival in vitro by affecting supportive cells, such as nurse-like cells, 8 found in the microenvironment of lymphoid tissues. Lenalidomide also can enhance T-cell proliferation 1 and interferon-g production 9 in response to CD3-crosslinking in vitro and dendritic-cell-mediated activation of T cells. 10 Moreover, lenalidomide can reverse noted functional defects of T cells in patients with CLL. 11,12 Finally, lenalidomide can also induce CLL B cells to express higher levels of immunostimulatory molecules such as CD80, CD86, HLA-DR, CD95, and CD40 in vitro, 5,13 thereby potentially enhancing their capacity to engage T cells in cognate interactions that lead to immune activation in response to leukemia-associated antigen(s). 14 However, lenalidomide may also have direct antiproliferative effects on CLL cells that account in part for its clinical activity in patients with this disease. This drug can inhibit proliferation of B-cell lymphoma lines 15 and induce growth arrest and apoptosis of mantlecell lymphoma cells. 16 Although originally considered an accumulative disease of resting G 0/1 lymphocytes, CLL increasingly is being reco...
Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid. We found that a humanized mAb specific for CD44 (RG7356) was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, RG7356 could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspasedependent apoptosis, independent of complement or cytotoxic effector cells. The cytotoxic effect of this mAb was not mitigated when the CLL cells were cocultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-μ. RG7356 induced rapid internalization of CD44 on CLL cells at 37°C, resulting in reduced expression of ZAP-70, which we found was complexed with CD44. Administration of this mAb at a concentration of 1 mg/kg to immune-deficient mice engrafted with human CLL cells resulted in complete clearance of engrafted leukemia cells. These studies indicate that this mAb might have therapeutic activity, particularly in patients with CLL that express ZAP-70.cell survival | preclinical studies | animal model | antibody therapy B -cell chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of mature, antigen-stimulated CD5+/ CD23+ B cells in blood, secondary lymphoid tissues, and marrow (1). Most of the circulating CLL cells in patients are arrested in the G0/G1 phase of the cell cycle and express high levels of antiapoptotic proteins (2). CLL therefore has been characterized as a process of defective apoptosis, rather than increased proliferation. However, despite their apparent longevity in vivo, CLL cells undergo spontaneous and drug-induced apoptosis in vitro, unless rescued by monocyte-derived Nurse-like cells (NLCs), follicular dendritic cells, or mesenchymal stromal cells (MSCs) (3-6). Thus, it has been postulated that CLL cells receive survival signals from these accessory cells, which constitute part of the CLL B-cell microenvironment in secondary lymphoid tissues and marrow (6). These survival signals can inhibit spontaneous or drug-induced apoptosis, particularly for CLL cells that express unmutated Ig heavy-chain variable genes (IGHVs) and/or the zeta-associated protein of 70 kDa (ZAP-70), which typically is not expressed by normal B cells (7). Patients with leukemia cells that possess such characteristics typically have a relatively short interval from diagnosis to initial therapy compared with patients with CLL cells that express mutated IGHVs or that lack expression of .One of the survival signals received by leukemia cells may be mediated via CD44, a surface glycoprotein receptor for the nonsulfated glycosaminoglycan hyaluronic acid (HA), which typically is found in the microenvironment of lymphoid tissues (13). CLL cells express high levels of CD44, particularly those that express unmutated IGHVs and/or . Upon binding HA in the extracellular matrix, CD44 activates the phosphoinositol 3-kinase (PI3K)/AKT and MAPK/ERK ...
We have previously shown that sorafenib, a multikinase inhibitor, exhibits cytotoxic effects on chronic lymphocytic leukemia (CLL) cells. Because the cellular microenvironment can protect CLL cells from drug-induced apoptosis, it is important to evaluate the effect of novel drugs in this context. Here we characterized the in vitro cytotoxic effects of sorafenib on CLL cells and the underlying mechanism in the presence of marrow stromal cells (MSCs) and nurselike cells (NLCs). One single dose of 10 μmol/L or the repeated addition of 1 μmol/L sorafenib caused caspase-dependent apoptosis and reduced levels of phosphorylated B-RAF, C-RAF, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia sequence 1 (Mcl-1) in CLL cells in the presence of the microenvironment. We show that the RAF/mitogen-activated protein kinase kinase (MEK)/ERK pathway can modulate Mcl-1 expression and contribute to CLL cell viability, thereby associating sorafenib cytotoxicity to its impact on RAF and Mcl-1. To evaluate if the other targets of sorafenib can affect CLL cell viability and contribute to sorafenib-mediated cytotoxicity, we tested the sensitivity of CLL cells to several kinase inhibitors specific for these targets. Our data show that RAF and vascular endothelial growth factor receptor (VEGFR) but not KIT, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) are critical for CLL cell viability. Taken together, our data suggest that sorafenib exerts its cytotoxic effect likely via inhibition of the VEGFR and RAF/MEK/ERK pathways, both of which can modulate Mcl-1 expression in CLL cells. Furthermore, sorafenib induced apoptosis of CLL cells from fludarabine refractory patients in the presence of NLCs or MSCs. Our results warrant further clinical exploration of sorafenib in CLL.
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