Despite 35 years of clinical trials, there is little improvement in one-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for c-Abl and Arg in melanoma progression. We demonstrate that the kinase activities of c-Abl and Arg (c-Abl, Arg) are elevated in primary melanomas (60%), in a subset of benign nevi (33%), and in some human melanoma cell lines. Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is required for melanoma cell proliferation, survival, and invasion. Significantly, we identify the mechanism by which activated c-Abl promotes melanoma invasion by showing that it transcriptionally upregulates MMP-1, and using rescue approaches we demonstrate that c-Abl promotes invasion via a STAT3→MMP-1 pathway. Additionally, we show that c-Abl and Arg are not merely redundant, as active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Most importantly, c-Abl and Arg not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg kinase activity with the c-Abl/Arg inhibitor, nilotinib, dramatically inhibits metastasis in a mouse model. Taken together, these data identify c-Abl and Arg as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may useful in preventing metastasis in patients with melanomas harboring active c-Abl and Arg.

Metastasis suppressors comprise a growing class of genes whose downregulation triggers metastatic progression. In contrast to tumor suppressors, metastasis suppressors are rarely mutated or deleted, and little is known regarding the mechanisms by which their expression is downregulated. Here, we demonstrate that the metastasis suppressor, NM23-H1, is degraded by lysosomal cysteine cathepsins (L,B), which directly cleave NM23-H1. In addition, activation of c-Abl and Arg oncoproteins induces NM23-H1 degradation in invasive cancer cells by increasing cysteine cathepsin transcription and activation. Moreover, c-Abl activates cathepsins by promoting endosome maturation, which facilitates trafficking of NM23-H1 to the lysosome where it is degraded. Importantly, the invasion- and metastasis-promoting activity of c-Abl/Arg is dependent on their ability to induce NM23-H1 degradation, and the pathway is clinically relevant as c-Abl/Arg activity and NM23-H1 expression are inversely correlated in primary breast cancers and melanomas. Thus, we demonstrate a novel mechanism by which cathepsin expression is upregulated in cancer cells (via Abl kinases). We also identify a novel role for intracellular cathepsins in invasion and metastasis (degradation of a metastasis suppressor). Finally, we identify novel crosstalk between oncogenic and metastasis suppressor pathways, thereby providing mechanistic insight into the process of NM23-H1 loss, which may pave the way for new strategies to restore NM23-H1 expression and block metastatic progression.

The incidence of melanoma is increasing, particularly in young women, and the disease remains incurable for many because of its aggressive, metastatic nature and its high rate of resistance to conventional, targeted, and immunological agents. Cathepsins are proteases that are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave or degrade proteins that restrict cancer progression, whereas extracellular cathepsins directly cleave the extracellular matrix and activate proinvasive proteases in the tumor microenvironment. Cathepsin secretion is markedly increased in cancer cells. We investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells. We found that the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) promoted the secretion of cathepsin B and cathepsin L by activating transcription factors (namely, Ets1, Sp1, and NF-κB/p65) that have key roles in the epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance. In some melanoma cell lines, Abl/Arg promoted the Ets1/p65-induced secretion of cathepsin B and cathepsin L in a kinase-independent manner, whereas in other melanoma lines, Abl/Arg promoted the kinase-dependent, Sp1/Ets1/p65-mediated induction of cathepsin L secretion and the Sp1/p65-mediated induction of cathepsin B secretion. As an indication of clinical relevance, the abundance of mRNAs encoding Abl/Arg, Sp1, Ets1, and cathepsins was positively correlated in primary melanomas, and Abl/Arg-driven invasion in culture and metastasis in vivo required cathepsin secretion. These data suggest that drugs targeting Abl kinases, many of which are FDA-approved, might inhibit cathepsin secretion in some melanomas and potentially other aggressive cancers harboring activated Abl kinases.

Previously, we demonstrated that Abl kinases are highly active in invasive breast cancer cell lines, and contribute to survival in response to nutrient deprivation, invasion and proliferation. To determine whether an Abl kinase inhibitor, STI571 (Gleevec; imatinib mesylate) sensitizes breast cancer cells to chemotherapeutic agents, we treated three breast cancer cell lines (BT-549, MDA-MB-231, and MDA-MB-468) that have active Abl kinases, with STI571 in combination with several conventional chemotherapeutic drugs frequently used to treat breast cancer, and assessed the effect on cell viability, proliferation, and apoptosis. We found that STI571 had synergistic effects with cisplatin in BT-549 and to some extent in MDA-MB-468 cells, STI571 synergized with camptothecin using an alternate dosing regimen in MDA-MB-231 cells, and STI571 synergistically sensitized MDA-MB-468 cells to paclitaxel and to high doses of 5-fluorouracil. Significantly, STI571 increased the ability of cisplatin to inhibit constitutive activation of PI3K/Akt, synergized with camptothecin to increase the stability of IκB in MDA-MB-231 cells, and in MDA-MB-468 cells, camptothecin and 5-fluorouracil inhibited STI571-dependent activation of STAT3. In other cell line/drug combinations, STI571 had additive or antagonistic effects, indicating that the ability of STI571 to sensitize breast cancer cells to chemotherapeutic agents is cell type-dependent. Significantly, unlike cisplatin, paclitaxel, and camptothecin, mechloroethamine was strongly antagonistic to STI571, and the effect was not cell line-dependent. Taken together, these data indicate that the cellular milieu governs the response of breast cancer cells to STI571/chemotherapeutic combination regimens, which suggests that treatment with these combinations requires individualization.

Despite 40 years of clinical trials, there has been little improvement in one-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for Abl kinases in melanoma progression. We demonstrate that Abl kinases (c-Abl, Arg) are activated in human melanoma cell lines, in primary melanomas (60%), and in a subset of benign nevi (33%). Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is required for melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. Moreover, active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3-MMP-1 pathway. Conversely, active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Paradoxically, c-Abl and Arg also increase expression of TIMP-1, a negative regulator of MMPs, both at the transcript and protein level via a STAT3-dependent pathway. In addition to negatively regulating MMPs, TIMPs also have anti-apoptotic functions, and high TIMP-1 expression is correlated with decreased survival and a poor clinical outcome in breast and colon cancer patients. Currently, we are in the process of testing whether c-Abl and Arg promote survival via induction of TIMP-1. Most importantly, we found that Abl kinases not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg with nilotinib dramatically inhibits lung colonization/metastasis in a mouse model using two different melanoma cell lines. Taken together, these data identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may useful in preventing metastasis in patients with melanomas harboring active Abl kinases. Citation Format: Sourik S. Ganguly, Leann S. Fiore, Jonathan T. Sims, J Woodrow Friend, DivyaMani Srinivasan, Matthew Thacker, Michael L. Cibull, Wang Chi, Rina Plattner. c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion, proliferation, survival, and drive metastatic progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C51.