Potential protein markers for breast cancer recurrence: a retrospective cohort study.

BACKGROUND: We evaluated five key proteins involved in various cancer-related pathways and assessed their relation to breast cancer recurrence. METHODS: We used the Kentucky Cancer Registry to retrospectively identify primary invasive breast cancer cases (n = 475) that were diagnosed and treated at University of Kentucky Medical Center between 2000 and 2007. Breast cancer recurrence was observed in 62 cases during the 5-year follow-up after diagnosis. Protein expression or activity level was analyzed from surgery tissue using immuno-histochemical assays. RESULTS: Compared to ER+/PR+/HER2- patients without recurrence, those with recurrence had higher TWIST expression (p = 0.049) but lower ABL1/ABL2 activity (p = 0.003) in primary tumors. We also found that triple-negative breast cancer patients with recurrence had higher SNAI1 expression compared to those without recurrence (p = 0.03). After adjusting for potential confounders, the higher ABL1/ABL2 activity in primary tumors was associated with a decreased risk of recurrence (OR 0.72, 95% CI 0.85-0.90) among ER+/PR+/HER2- patients. In addition, among patients with recurrence we observed that the activity level of ABL1/ABL2 was significantly increased in recurrent tumors compared to the matched primary tumors regardless of the subtype (p = 0.013). CONCLUSIONS: These findings provide evidence that the expression/activity level of various proteins may be differentially associated with risk of recurrence of breast tumor subtypes.

ABL1/2 and DDR1 Drive MEKi Resistance in NRAS-Mutant Melanomas by Stabilizing RAF/MYC/ETS1 and Promoting RAF Homodimerization.

Melanomas harboring NRAS mutations are a particularly aggressive and deadly subtype. If patients cannot tolerate or the melanomas are insensitive to immune checkpoint blockade, there are no effective 2nd-line treatment options. Drugs targeting the RAF/MEK/ERK pathway, which are used for BRAF-mutant melanomas, do little to increase progression-free survival (PFS). Here, using both loss-of-function and gain-of-function approaches, we show that ABL1/2 and DDR1 are critical nodes during NRAS-mutant melanoma intrinsic and acquired MEK inhibitor (MEKi) resistance. In some acquired resistance cells, ABL1/2 and DDR1 cooperate to stabilize RAF proteins, activate ERK cytoplasmic and nuclear signaling, repress p27/KIP1 expression, and drive RAF homodimerization. In contrast, other acquired resistance cells depend solely on ABL1/2 for their survival, and are sensitive to highly specific allosteric ABL1/2 inhibitors, which prevent ß-catenin nuclear localization and destabilize MYC and ETS1 in an ERK-independent manner. Significantly, targeting ABL1/2 and DDR1 with an FDA-approved anti-leukemic drug, reverses intrinsic MEKi resistance, delays acquisition of acquired resistance, and doubles the survival time in a NRAS-mutant mouse model. These data indicate that repurposing FDA-approved drugs targeting ABL1/2 and DDR1 may be a novel and effective strategy for treating patients with treatment-refractory NRAS-driven melanomas.

Inhibition of ABL1 by tyrosine kinase inhibitors leads to a downregulation of MLH1 by Hsp70-mediated lysosomal protein degradation.

The DNA mismatch repair (MMR) pathway and its regulation are critical for genomic stability. Mismatch repair (MMR) follows replication and repairs misincorporated bases and small insertions or deletions that are not recognized and removed by the proofreading polymerase. Cells deficient in MMR exhibit an increased overall mutation rate and increased expansion and contraction of short repeat sequences in the genome termed microsatellite instability (MSI). MSI is often a clinical measure of genome stability in tumors and is used to determine the course of treatment. MMR is also critical for inducing apoptosis after alkylation damage from environmental agents or DNA-damaging chemotherapy. MLH1 is essential for MMR, and loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. In this study, we report that tyrosine kinase inhibitors, imatinib and nilotinib, lead to decreased MLH1 protein expression but not decreased MLH1 mRNA levels. Of the seven cellular targets of Imatinib and nilotinib, we show that silencing of ABL1 also reduces MLH1 protein expression. Treatment with tyrosine kinase inhibitors or silencing of ABL1 results in decreased apoptosis after treatment with alkylating agents, suggesting the level of MLH1 reduction is sufficient to disrupt MMR function. We also report MLH1 is tyrosine phosphorylated by ABL1. We demonstrate that MLH1 downregulation by ABL1 knockdown or inhibition requires chaperone protein Hsp70 and that MLH1 degradation can be abolished with the lysosomal inhibitor bafilomycin. Taken together, we propose that ABL1 prevents MLH1 from being targeted for degradation by the chaperone Hsp70 and that in the absence of ABL1 activity at least a portion of MLH1 is degraded through the lysosome. This study represents an advance in understanding MMR pathway regulation and has important clinical implications as MMR status is used in the clinic to inform patient treatment, including the use of immunotherapy.

A new link between the c-Abl tyrosine kinase and phosphoinositide signalling through PLC-gamma1.

The c-Abl tyrosine (Tyr) kinase is activated after platelet-derived-growth factor receptor (PDGFR) stimulation in a manner that is partially dependent on Src kinase activity. However, the activity of Src kinases alone is not sufficient for activation of c-Abl by PDGFR. Here we show that functional phospholipase C-gamma1 (PLC-gamma1) is required for c-Abl activation by PDGFR. Decreasing cellular levels of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by PLC-gamma1-mediated hydrolysis or dephosphorylation by an inositol polyphosphate 5-phosphatase (Inp54) results in increased Abl kinase activity. c-Abl functions downstream of PLC-gamma1, as expression of kinase-inactive c-Abl blocks PLC-gamma1-induced chemotaxis towards PDGF-BB. PLC-gamma1 and c-Abl form a complex in cells that is enhanced by PDGF stimulation. After activation, c-Abl phosphorylates PLC-gamma1 and negatively modulates its function in vivo. These findings uncover a newly discovered functional interdependence between non-receptor Tyr kinase and lipid signalling pathways.

KEAP1 Is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer.

Artesunate is the most common treatment for malaria throughout the world. Artesunate has anticancer activity likely through the induction of reactive oxygen species, the same mechanism of action utilized in Plasmodium falciparum infections. Components of the kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which regulates cellular response to oxidative stress, are mutated in approximately 30% of non-small-cell lung cancers (NSCLC); therefore, we tested the hypothesis that KEAP1 is required for artesunate sensitivity in NSCLC. Dose response assays identified A549 cells, which have a G333C-inactivating mutation in KEAP1, as resistant to artesunate, with an IC50 of 23.6 µM, while H1299 and H1563 cells were sensitive to artesunate, with a 10-fold lower IC50. Knockdown of KEAP1 through siRNA caused increased resistance to artesunate in H1299 cells. Alternatively, the pharmacological inhibition of NRF2, which is activated downstream of KEAP1 loss, by ML385 partially restored sensitivity of A549 cells to artesunate, and the combination of artesunate and ML385 was synergistic in both A549 and H1299 cells. These findings demonstrate that KEAP1 is required for the anticancer activity of artesunate and support the further development of NRF2 inhibitors to target patients with mutations in the KEAP1/NRF2 pathway.

Combating acquired resistance to MAPK inhibitors in melanoma by targeting Abl1/2-mediated reactivation of MEK/ERK/MYC signaling.

Metastatic melanoma remains an incurable disease for many patients due to the limited success of targeted and immunotherapies. BRAF and MEK inhibitors reduce metastatic burden for patients with melanomas harboring BRAF mutations; however, most eventually relapse due to acquired resistance. Here, we demonstrate that ABL1/2 kinase activities and/or expression are potentiated in cell lines and patient samples following resistance, and ABL1/2 drive BRAF and BRAF/MEK inhibitor resistance by inducing reactivation of MEK/ERK/MYC signaling. Silencing/inhibiting ABL1/2 blocks pathway reactivation, and resensitizes resistant cells to BRAF/MEK inhibitors, whereas expression of constitutively active ABL1/2 is sufficient to promote resistance. Significantly, nilotinib (2nd generation ABL1/2 inhibitor) reverses resistance, in vivo, causing prolonged regression of resistant tumors, and also, prevents BRAFi/MEKi resistance from developing in the first place. These data indicate that repurposing the FDA-approved leukemia drug, nilotinib, may be effective for prolonging survival for patients harboring BRAF-mutant melanomas.

Activation of Abl tyrosine kinases promotes invasion of aggressive breast cancer cells.

The Abl family of nonreceptor tyrosine kinases consists of two related proteins, c-Abl and Abl-related gene (Arg). Activated forms of the Abl kinases (BCR-Abl, Tel-Abl, and Tel-Arg) induce the development of human leukemia; it is not known, however, whether Abl kinases are activated in solid tumors or whether they contribute to tumor development or progression. Previously, we showed that Abl kinases are activated downstream of growth factor receptors, Src family kinases, and phospholipase Cgamma1 (PLCgamma1) in fibroblasts and influence growth factor-mediated proliferation, membrane ruffling, and migration. Growth factor receptors, Src kinases, and PLCgamma1 are deregulated in many solid tumors and drive tumor invasion and metastasis. In this study, we found that Abl kinases are constitutively activated, in highly invasive breast cancer cell lines, downstream of deregulated ErbB receptors and Src kinases. Furthermore, activation of Abl kinases promotes breast cancer cell invasion, as treatment of cells with the Abl kinase inhibitor, STI571, or silencing c-Abl and Arg expression with RNA interference dramatically inhibits Matrigel invasion. This is the first evidence that (a) Abl kinases are deregulated and activated in a nonhematopoietic cancer, (b) activation of Abl kinases in breast cancer cells occurs via a novel mechanism, and (c) constitutive activation of Abl kinases promotes invasion of breast cancer cells. These data suggest that pharmacologic inhibitors targeted against Abl kinases could potentially be useful in preventing breast cancer progression in tumors harboring activated Abl kinases.

EnABLing Cathepsin-Driven Melanoma Metastasis.

Metastatic melanoma remains incurable for many due to its aggressive nature. Secreted cathepsins promote metastasis by cleaving matrix and activating pro-invasive proteases. We reported that ABL kinases induce cathepsin secretion and subsequent metastasis by activating ETS1, SP1, and RELA pathways, indicating that ABL inhibitors may serve as novel anti-cathepsin agents.

EnABLing Tumor Growth and Progression: Recent progress in unraveling the functions of ABL kinases in solid tumor cells.

PURPOSE OF REVIEW: The goal of this review is to summarize our current knowledge regarding how ABL family kinases are activated in solid tumors and impact on solid tumor development/progression, with a focus on recent advances in the field. RECENT FINDINGS: Although ABL kinases are known drivers of human leukemia, emerging data also implicates the kinases in a large number of solid tumor types where they promote diverse processes such as proliferation, survival, cytoskeletal reorganization, cellular polarity, EMT (epithelial-mesenchymal-transition), metabolic reprogramming, migration, invasion and metastasis via unique signaling pathways. ABL1 and ABL2 appear to have overlapping but also unique roles in driving these processes. In some tumor types, the kinases may act to integrate pro- and anti-proliferative and -invasive signals, and also may serve as a switch during EMT/MET (mesenchymal-epithelial) transitions. CONCLUSIONS: Most data indicate that targeting ABL kinases may be effective for reducing tumor growth and preventing metastasis; however, ABL kinases also may have a tumor suppressive role in some tumor types and in some cellular contexts. Understanding the functions of ABL kinases in solid tumors is critical for developing successful clinical trials aimed at targeting ABL kinases for the treatment of solid tumors.

Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis.

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.

Bidirectional signaling links the Abelson kinases to the platelet-derived growth factor receptor.

The c-Abl nonreceptor tyrosine kinase is activated by growth factor signals such as the platelet-derived growth factor (PDGF) and functions downstream of the PDGF-beta receptor (PDGFR) to mediate biological processes such as membrane ruffling, mitogenesis, and chemotaxis. Here, we show that the related kinase Arg is activated downstream of PDGFRs in a manner dependent on Src family kinases and phospholipase C gamma1 (PLC-gamma1)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, as we showed previously for c-Abl. PIP2, a highly abundant phosphoinositide known to regulate cytoskeletal and membrane proteins, inhibits the tyrosine kinase activities of both Arg and c-Abl in vitro and in cells. We now demonstrate that c-Abl and Arg form inducible complexes with and are phosphorylated by the PDGFR tyrosine kinase in vitro and in vivo. Moreover, c-Abl and Arg, in turn, phosphorylate the PDGFR. We show that c-Abl and Arg exhibit nonredundant functions downstream of the activated PDGFR. Reintroduction of c-Abl into Arg-Abl double-null fibroblasts rescues the ability of PLC-gamma1 to increase PDGF-mediated chemotaxis, while reexpression of Arg fails to rescue the chemotaxis defect. These data show that, although both kinases are activated and form complexes with proteins in the PDGFR signaling pathway, only c-Abl functions downstream of PLC-gamma1 to mediate chemotaxis.

c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer.

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.

CD151-α3ß1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling.

Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3ß1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3ß1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of ß-catenin and Axin-2 as well as resistance to the inhibition in ß-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3ß1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling.

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.

Activation of abl family kinases in solid tumors.

Although c-Abl and Arg non-receptor tyrosine kinases are well known for driving leukemia development, their role in solid tumors has not been appreciated until recently. Accumulating evidence now indicates that c-Abl and/or Arg are activated in some solid tumor cell lines via unique mechanisms that do not involve gene mutation/translocation, and c-Abl/Arg activation promotes matrix degradation, invasion, proliferation, tumorigenesis, and/or metastasis, depending on the tumor type. However, some data suggest that c-Abl also may suppress invasion, proliferation, and tumorigenesis in certain cell contexts. Thus, c-Abl/Arg may serve as molecular switches that suppress proliferation and invasion in response to some stimuli (e.g., ephrins) or when inactive/regulated, or as promote invasion and proliferation in response to other signals (e.g., activated growth factor receptors, loss of inhibitor expression), which induce sustained activation. Clearly, more data are required to determine the extent and prevalence of c-Abl/Arg activation in primary tumors and during progression, and additional animal studies are needed to substantiate in vitro findings. Furthermore, c-Abl/Arg inhibitors have been used in numerous solid tumor clinical trials; however, none of these trials were restricted to patients whose tumors expressed highly activated c-Abl/Arg (targeted trial). Targeted trials are critical for determining whether c-Abl/Arg inhibitors can be effective treatment options for patients whose tumors are driven by c-Abl/Arg.

Enhanced resistance to tamoxifen by the c-ABL proto-oncogene in breast cancer.

Targeting the estrogen receptor is an important strategy in breast cancer therapy. However, although inhibiting estrogen receptor function with specific estrogen receptor modulators can achieve a primary response in cancer patients, intrinsic or subsequently acquired resistance to the therapy remains a major obstacle in the clinic. Thus, it is critical to gain a more thorough understanding of how estrogen receptor functions are regulated in breast cancer.Here, we demonstrate that the non-receptor tyrosine kinase c-ABL is a functional partner of the estrogen receptor, as expression of c-ABL sustained transcriptional activity of the estrogen receptor. More importantly, inhibition of c-ABL resulted in sensitization to treatment by tamoxifen (TAM) in estrogen receptor-positive breast cancer cells, as manifested by inhibition of cell survival and suppression of anchorage-independent growth. We found that c-ABL interacts with estrogen receptor in breast cancer cells and that expression of c-ABL is a frequent event in primary breast cancer tumor tissues. In estrogen receptor-positive tumors, the expression of c-ABL significantly correlated with disease progression and metastasis. This study shows that c-ABL regulates the cellular response to TAM through functional interaction with the estrogen receptor, which suggests c-ABL as a therapeutic target and a prognostic tumor marker for breast cancer.

MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines.

PURPOSE: This study will determine whether MTT assays accurately assess the effect of STI571 (Gleevec; Abl kinase inhibitor) on the viability of cancer cells containing highly active Abl kinases. METHODS: Growth kinetics, tritiated thymidine, fluorescent caspase, MTT, and Cell Titer Glo (CTG) assays were used to determine the effect of STI571 on growth, proliferation, apoptosis, and viability of melanoma and breast cancer cells. RESULTS: STI571 inhibited growth and proliferation, and increased apoptosis. However, MTT assays indicated that STI571 increased cell viability. In contrast, STI571 induced a dose-dependent decrease in viability using CTG assays. CONCLUSIONS: Doses of STI571 (1-10 microM) required to inhibit endogenous Abl kinases interfere with the MTT assay, and therefore MTT cannot be used to determine the effect of STI571 on viability using these doses. Additionally, caution should be utilized when interpreting the results of MTT assays used to screen kinase inhibitors for anti-cancer activity, as drug effectiveness may be minimized.

Reciprocal regulation of Abl and receptor tyrosine kinases.

Previously, we showed that Abl kinases (c-Abl, Arg) are activated downstream of PDGF in a manner dependent on Src kinases and PLC-gamma1, and promote PDGF-mediated proliferation and migration of fibroblasts. We additionally demonstrated that Abl kinases bind directly to PDGFR-beta via their SH2 domains.In this study, we extend these findings by demonstrating that Abl kinases also are activated downstream of aPDGF autocrine growth loop in glioblastoma cells, indicating that the PDGFR-Abl signaling pathway also is likely to be important in glioblastoma development and/or progression.We recently showed that Abl kinases are highly active in many breast cancer cell lines, and the Her-2 receptor tyrosine kinase contributes to c-Abl and Arg kinase activation. In this study, we show that Abl kinase SH2 domains bind directly to Her-2, and like PDGFR-beta , Her-2 directly phosphorylates c-Abl. Previously, we demonstrated that PDGFR-beta directly phosphorylates Abl kinases in vitro, and Abl kinases reciprocally phosphorylate PDGFR-beta . Here, we show that PDGFR-beta-phosphorylation of Abl kinases has functional consequences as PDGFR-beta phosphorylates Abl kinases on Y245 and Y412, sites known to be required for activation of Abl kinases. Moreover, PDGFR-beta phosphorylates Arg on two additional unique sites whose function is unknown. Importantly, we also show that Abl-dependent phosphorylation of PDGFR-beta has functional and biological significances. c-Abl phosphorylates three tyrosine residues on PDGFR-beta (Y686, Y934, Y970), while Arg only phosphorylatesY686. Y686 and Y934 reside in PDGFR-beta catalytic domains, while Y970 is in the C-terminal tail. Using site-directed mutagenesis, we show that Abl-dependent phosphorylation of PDGFR-beta activates PDGFR-beta activity, in vitro, but serves to downregulate PDGFR-mediated chemotaxis. These data are exciting as they indicate that Abl kinases not only are activated by PDGFR and promote PDGFR-mediated proliferation and migration,but also act in an intricate negative feedback loop to turn-off PDGFR-mediated chemotaxis.

STI571 sensitizes breast cancer cells to 5-fluorouracil, cisplatin and camptothecin in a cell type-specific manner.

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; 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 in BT-549 cells, synergized with camptothecin to increase the stability of IkappaB 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.