MOLECULAR MECHANISMS BY WHICH c-ABL AND ARG MEDIATE MELANOMA INVASION AND METASTASIS

Metastasis is one of the main causes of death in cancer patients. Metastatic melanoma is a death sentence, as chemotherapeutic agents have a 5% success rate or do not extend survival beyond 10 months. The lack of effective chemotherapeutic agents for treating metastatic melanoma indicates a dire need to identify new drug targets and develop new therapies. Our lab has previously shown that the kinase activity of Abelson family of non-receptor tyrosine kinases (c-Abl and Arg) is elevated in invasive breast cancer cell lines as compared to non-invasive cell lines. Previous studies from our lab have shown that Abl kinases are convergent point of ErbB2 and Src Kinases in melanoma cells and Abl kinases promote invasion by an undefined mechanism. Although Abl kinases promote invasion, it is not known whether they are important for metastastic potential. For the first time, we report that Abl kinases promote melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. To investigate the mechanism by which Abl kinases promote invasion, we found out that active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3àMMP-1 pathway. In contrast, active Arg drives invasion in a STAT3-independent manner, and upregulates the expression of MMP-3 and MT1-MMP, in addition to MMP-1. We also found that Abl kinases promote invasion via lysosomal degradation of a metastasis suppressor, NM23-H1 by activating lysosomal cathepsins B and L, which directly cleave and degrade NM23-H1. Furthermore, c-Abl and Arg are activated in primary melanomas and cAbl/Arg activity is inversely correlated with NM23-H1 expression both in primary melanoma and human melanoma cells. We also demonstrate, for the first time that active Abl kinases 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. In summary, we identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and our data indicate that nilotinib may be useful in preventing metastasis in a select group of patients, harboring active Abl kinases

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis

Global deletion of tetraspanin CD82 attenuates bone growth and enhances bone marrow adipogenesis

CD82 is a widely expressed member of the tetraspanin family of transmembrane proteins known to control cell signaling, adhesion, and migration. Tetraspanin CD82 is induced over 9-fold during osteoclast differentiation in vitro; however, its role in bone homeostasis is unknown. A globally deleted CD82 mouse model was used to assess the bone phenotype. Based on microCT and 4-point bending tests, CD82-deficient bones are smaller in diameter and weaker, but display no changes in bone density. Histomorphometry shows a decrease in size, erosion perimeter, and number of osteoclasts in situ, with a corresponding increase in trabecular surface area, specifically in male mice. Male-specific alterations are observed in trabecular structure by microCT and in vitro differentiated osteoclasts are morphologically abnormal. Histomorphometry did not reveal a significant reduction in osteoblast number; however, dynamic labeling reveals a significant decrease in bone growth. Consistent with defects in OB function, OB differentiation and mineralization are defective in vitro, whereas adipogenesis is enhanced. There is a corresponding increase in bone marrow adipocytes in situ. Thus, combined defects in both osteoclasts and osteoblasts can account for the observed bone phenotypes, and suggests a role for CD82 in both bone mesenchyme and myeloid cells.Van Andel Research Institute; Van Andel Institute Graduate School12 month embargo; published online: 18 May 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

<div><p>Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.</p> </div

Mechanisms by which imatinib abrogates intrinsic doxorubicin resistance.

<p>Imatinib prevents activation of STAT3, which promotes doxorubicin-mediated NF-κB phosphorylation and nuclear translocation and NF-κB-mediated inhibition of expression of anti-apoptotic proteins such as cIAP1/XIAP. Imatinib-mediated inhibition of STAT3 also prevents activation of a HSP27/p38/Akt pro-survival pathway.</p

Resistance to doxorubicin occurs via ABCB1-dependent and ABCB1-independent mechanisms.

<p>(<b>A, B</b>) ABCB1 was inhibited with verapamil (<b>A</b>) or silenced with siRNA (<b>B</b>), cells were treated with imatinib (10 µM) and/or doxorubicin (0.5 µM) for 72 h, and cell viability assessed. Mean±SEM from 3 independent experiments. (A) Dox+imatinib, CI = 0.66±0.19; Dox+verapamil, CI = 0.14±0.04; Dox+imatinib+verapamil, CI = 0.47±0.5. Representative Western blot (right). Some error bars are too small to visualize.</p

Androgen receptor-induced integrin α6β1 and Bnip3 promote survival and resistance to PI3K inhibitors in castration-resistant prostate cancer

The androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins alpha 6 beta 1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin alpha 6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin alpha 6 beta 1-dependent nuclear translocation of HIF1 alpha. Integrins alpha 6 beta 1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin alpha 6 beta 1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.6 month embargo; published online: 21 June 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Imatinib abrogates doxorubicin resistance, in part, by increasing p65 nuclear translocation, and inhibiting expression of NF-κB targets.

<p>(<b>A</b>) Nuclear lysates from doxorubin (0.5 µM-top; 1 µM-bottom)/imatinib (10 µM)-treated 435s/M14 cells (8 h) were blotted. Graphs are Mean±SEM from 3 independent experiments. (<b>B</b>) Parental (435s/M14) and doxorubicin-resistant (435s/M14-DR) cells, stably expressing a 3X-NF-κB-luciferase reporter, were drug treated (8 h), and luciferase activity assessed. Graphs are Mean±SEM from 3 independent experiments. (<b>C, D</b>) Lysates from treated cells were analyzed by Western blot (40 h treatment). Representative blots are shown. Mean±SEM from 3 independent experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055509#pone.0055509.s006" target="_blank">Figure S6D,E</a>. (<b>E</b>) 435s/M14 cells transfected with p65 siRNA were drug-treated (8 h), and PARP and caspase-3 cleavage assessed. Graphs are Mean±SEM from 3 independent experiments. *<i>p</i><0.05, **<i>p</i>≤0.01 (see methods).</p

Imatinib abrogates doxorubicin resistance, in part, by preventing activation of a STAT3/HSP27/p38/Akt survival pathway.

<p>(<b>A, B</b>) 435s/M14 cells were treated with imatinib (10 µM) (<b>A</b>) or transfected with STAT3 siRNA (<b>B</b>), treated with doxorubicin (p38, Akt, 0.5 µM; HSP27, 1 µM) for 40 h, and lysate from attached/detached cells blotted with antibodies. Experiments were performed 3 times; representative blots are shown. Mean±SEM for 3 experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055509#pone.0055509.s007" target="_blank">Figure S7A</a>. (<b>C</b>) 435s/M14 cells expressing pcDNA or STAT3C were drug treated (40 h), and lysate from attached/detached cells were blotted with antibodies. Graphs are Mean±SEM from 3 independent experiments (bottom). (<b>D</b>) 435s/M14 cells expressing vector or constitutively active PI3K (E545K) were drug-treated (8 h) and PARP cleavage assessed. Graph is Mean±SEM from 3 independent experiments. Representative Western blot (right). For all subfigures, some error bars are too small to visualize. *<i>p</i><0.05, **<i>p</i>≤0.01, ***p<0.001 (see methods).</p

Imatinib reverses doxorubicin resistance by inhibiting cell survival.

<p>Caspase-3/7 activity (<b>A,B,E</b>), Annexin V staining (<b>C</b>), or PARP cleavage (<b>D,F</b>) were assessed in lysates from attached and detached treated cells (40 h unless otherwise indicated). Mean±SEM of 3 experiments (<b>A,B,E</b>) or representative of 3 independent experiments (<b>C,D,F</b>). Some error bars (A,B,E) are too small to visualize. *p<0.05, **<i>p</i>≤0.01, ***<i>p</i><0.001 (see methods).</p