Dasatinib-sensitive Cells Research Articles

Abstract 1681: The alternative mechanism of Src inhibition in colon cancer cells

Abstract In colon cancer, Src showed the high expression in tumor cells and was known as an important migration marker that plays the role in coordinate with many important cellular signal pathways. According to previous studies, cell viability and migration ability can be inhibited by Src inhibitor, Dasatinib. However, the clinical study demonstrated the survival rate has no significant increase in Dasatinib treatment. Therefore, we want to figure out what is the real function of Src in colon cancer cell in this study. HT-29, colon cancer cell with epithelial cell morphology was used in the study. After treated with Dasatinib, cells were followed by reattachment experiment. Then the cell growth, migration ability, signaling pathway transition, molecular localization and expressions were analyzed. The results showed Src inhibition can reduce EGFR activation on cell-cell contacts and increased Akt phosphorylation upon EGF stimulation. We further found Dasatinib has limited effect on cell toxicity, however, can be the selector to separate two cell populations, Dasatinib sensitive (DSTS) and Dasatinib resistance (DSTR) cells, interestingly, the DSTS cells were grown as colony phenotype efficiently and majorly through EGFR/AKT pathway. Meanwhile, DSTR was demonstrated the promotion of collective cell migration ability by EGFR/Ecad pathway. The results of this study suggested Src activity plays novel role in switch EGFR/Akt and EGFR/Ecad pathways in colon cancer cells and harboring different strategies to survive, therefore, the alternative resistance mechanism of Src inhibitor in colon cancer cells may need be concerned in clinical application. Citation Format: Yi-Wen Lu, Xiang-Ling Hou, Yen-Tse Lu, Chia-Wen Lin, Chien-Chi Huang, Wei-Ting Chao. The alternative mechanism of Src inhibition in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1681.

Inhibition of Akt/mTOR pathway overcomes intrinsic resistance to dasatinib in triple-negative breast cancer

Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.

Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Transitory dasatinib-resistant states in KITmut t(8;21) acute myeloid leukemia cells correlate with altered KIT expression

KIT inhibition with dasatinib represents a promising approach to targeted therapy in t(8;21) acute myeloid leukemia (AML) and clinical trials are currently evaluating its clinical relevance. However, data on continuous long-term dasatinib exposure of AML cells are limited and the potential effects on KIT inhibition and dasatinib sensitivity are unexplored. Treatment-related resistance ultimately limits clinical efficacy of tyrosine kinase inhibitors (TKI), which could similarly apply to dasatinib in t(8;21) AML. In this study, we used the dasatinib-sensitive KIT(mut) t(8;21) AML cell line Kasumi-1 to model, in a confined and controllable way, molecular effects upon continuous dasatinib treatment. Long-term dasatinib exposure at clinically relevant levels resulted in markedly decreased drug-sensitivity of KIT(mut) t(8;21) AML cells. Notably, all dasatinib-resistant clones lacked secondary KIT-mutations. Instead, persistent growth correlated with alterations in KIT expression levels-that is, either KIT overexpression with maintained downstream signaling or KIT downregulation with concomitant activation of alternate pathways. Although KIT overexpression was associated with retained receptor activity and STAT3 activation, KIT downregulation correlated with decreased STAT3 levels and increased ERK-signaling. Importantly, brief discontinuation of dasatinib restored dasatinib-sensitivity associated with reversal of signaling signatures similar to treatment-naive, dasatinib-sensitive cells. The observed desensitization of KIT(mut) t(8;21) AML cells upon continuous dasatinib exposure suggests that therapy-related acquisition of resistance could pose significant limitations on therapeutic efficiency. Notably, we identified TKI-resistant states of transient nature that correlate with alterations in KIT expression and can be reversed upon brief inhibitor withdrawal. These findings indicate that discontinuing treatment maintains dasatinib sensitivity in KIT(mut) AML cells.

Src: a potential target for the treatment of triple-negative breast cancer

BackgroundTriple-negative breast cancers lack expression of estrogen and progesterone receptors and overexpression of human epidermal growth factor receptor 2 (HER2). Unlike other subgroups of patients with breast cancer, targeted therapy is currently unavailable for these patients. The aim of this study was to investigate v-src sarcoma viral oncogene homolog (Src) as a potential target for the treatment of triple-negative breast cancer. MethodsExpression of Src was measured in 87 triple-negative and 93 non-triple-negative breast cancers. Dasatinib (an inhibitor of Src) was tested in a panel of breast cancer cell lines. ResultsCytoplasmic expression of Src was detected in 83 (95%) triple-negative samples versus 78 (84%) non-triple-negative samples (P = 0.012), while membrane Src was detected in 78% triple-negative compared with 38% of non-triple-negative specimens (P < 0.0001). Dasatinib inhibited growth in three of five triple-negative cell lines (IC50 < 1 μM). Dasatinib combined with cisplatin was synergistic in the three dasatinib-sensitive cell lines (combination index < 0.9). Dasatinib, in combination with 5′-deoxy-5′-fluoruridine, displayed synergy or additivity. Moderate synergy was observed with docetaxel (Taxotere) in two cell lines but the combination was antagonistic in HCC-1143 cells. ConclusionsWe conclude that dasatinib with cisplatin is a rational drug combination for testing in triple-negative breast cancer.

Identification of predictive biomarkers for dasatinib treatment of metastatic melanoma.

8578 Background: Metastatic melanoma remains an almost certainly fatal disease. Current therapies fail to improve survival rates; therefore it is critical to identify alternate therapies for this disease. Dasatinib, a multi-target tyrosine kinase inhibitor, has anti-proliferative and anti-invasive effects in vitro in melanoma. However, clinical trials with dasatinib have demonstrated modest activity in metastatic melanoma. Identification of predictive biomarkers for dasatinib may facilitate selection of melanoma patients that are more likely to respond to dasatinib. Methods: We examined the effect of dasatinib on proliferation in 8 melanoma cell lines. We analyzed the expression of a previously identified 6-gene panel (ANXA1, CAV-1 CAV-2, EphA2, IGFBP2 and PTRF) of biomarkers in the panel of cell lines. We correlated response to dasatinib with expression of each gene at both the mRNA and protein level. ANXA1, CAV-1 and EphA2 expression were analyzed in 121 melanoma tumor samples by immunohistochemical (IHC) staining. Results: Dasatinib inhibits growth in 3 of the 8 melanoma cell lines tested. mRNA expression of the 6-gene markers did not correlate with response whilst higher protein expression of ANXA1 (p=0.04), CAV-1 (p=0.05) and EphA2 (p=0.02) correlated significantly with response to dasatinib in the panel of cell lines. Furthermore, the 3 dasatinib sensitive cell lines expressed high levels of all three markers. Although ANXA1 was detected in all of the resistant cell lines, CAV1 and EphA2 were only detected in 3 of the 5, and none of the resistant cell lines expressed high levels of all three markers. ANXA1 protein was detected in 82 % (97/119) of tumors, CAV-1 in 45 % (54/121) and EphA2 in 75 % (90/120). 34 % (38/112) of the melanoma specimens expressed all three markers and 12 % (13/112) demonstrated high levels of ANXA1, CAV-1 and EphA2 staining (2+ (moderate) or 3+ (strong) by IHC). Conclusions: High levels of ANXA1, CAV-1 and EphA2 correlate with response to dasatinib in melanoma cell lines. Immunohistochemical analysis suggests that 12% of melanomas (13/112) showed high levels of staining for the three markers. This subgroup may represent a population of patients who may derive clinical benefit from dasatinib treatment.

C-src: A potential target for the treatment of triple-negative breast cancer.

10617 Background: Triple-negative (TN) breast cancers are so-called as they lack expression ER and PR and overexpression of HER-2. Unlike other subgroups of patients with breast cancer, targeted therapy is currently unavailable for these patients. Consequently, the only systemic therapy for TN patients is chemotherapy. The aim of this study was to investigate the use of c-src as a potential target for the treatment of patients with TN breast cancer. Methods: Src immunoreactivity was determined using TMAs in 87 randomly-selected TN and 93 non-TN breast cancers diagnosed at St Vincent's Univ Hospital over the period 2002-2006. In vitro proliferation assays were performed to test combinations of dasatinib and cytotoxic drugs in breast cancer cell lines. Synergy analysis was performed using the Tchou and Talalay equation on CalcuSyn software. Results: 83/87 (95%) of TN cancers were positive for src compared to 78/93 (84%) of non-TN samples (p = 0.012, chi square test). Similarly, membrane staining for src was found more frequently in TN than in non-TN samples (74% vs 38%, p < 0.0001, chi square test). Using 9 different breast cancer cell lines in culture, dasatinib (an inhibitor of c-src) blocked growth in 3 out of 5 TN cell lines with IC50 < 1 μM (i.e, in MDA-MB-231, HCC-1143, HCC-1937) but in only 1 out of 4 HER2 amplified cell lines. Dasatinib combined with docetaxel displayed moderate synergy in MDA-MB-231 and HCC-1937 cells (CI < 0.9), but was antagonistic in HCC-1143 cells (CI > 1.1). Combined treatment with dasatinib and cisplatin was synergistic in the three dasatinib sensitive cell lines (CI < 0.9). In MDA-MB-231 and HCC-1143 cells, combined treatment with dasatinib and 5′-DFUR displayed synergy (CI < 0.9), whereas the combination was additive in HCC-1937 cells (CI = 0.98). Conclusions: We conclude that a combination of dasatinib and cisplatin is a potential new treatment for patients with TN-negative breast cancer. Acknowledgements: The authors wish to thank Science Foundation Ireland (08/SRC/B1410) and the Health Research Board (CSA/2007/1) for funding this work. No significant financial relationships to disclose.

Dual inhibition of αV integrins and Src kinase activity in colon cancer cells

e14609 Background: Integrins are commonly upregulated in tumor cells and are important regulators of invasion and metastasis. Integrin signaling is initiated upon engagement of ECM and requires Src as well as various other proteins including ILK, FAK, and paxillin. Methods: Presence of αv integrins on CRC cell lines was established by flow cytometry. CNTO 95 (10μg/ml, a monoclonal anti-αv integrin antibody; Ortho Biotech), and dasatinib (&lt;200nM; BMS, src small molecule inhibitor) were used to study the effects of combined integrin and src inhibition on proliferation and serum-induced migration of colon cancer cell lines in vitro. Downstream signaling changes were assessed by immunoblotting for phosphorylated forms of src, FAK (Y397, Y576/577 and Y925), paxillin, GSK3β and AKT (S473) in HT29, HCT116 and RKO. Results: Proliferation was inhibited by dasatinib in HT29, HCT116, DLD1 and HCT15 in a dose-dependent manner, while RKO and SW48 were resistant. Combining CNTO 95 with dasatinib further inhibited proliferation in all dasatinib-sensitive cells, yet resistant cells were unaffected. Migration was blocked by both CNTO 95 and dasatinib in all cell lines, and combining the two drugs produced augmented effect. Src activation and src- dependent FAK phosphorylation at sites 576 and 925 were blocked by dasatinib; CNTO 95 had little effect alone, but potentiated the effect of dasatinib. Paxillin phosphorylation was modestly blocked by both compounds, but the combination produced significantly augmented inhibition in the three cell lines tested. The phosphorylation status of AKT and GSK-3β, which are downstream of ILK, was inhibited by both drugs as single agents. The combination of dasatinib and CNTO 95 produced further inhibition. Conclusions: Dual inhibition of Src by dasatinib and αv integrins by CNTO 95 produced additive to synergistic inhibition of proliferation in dasatinib sensitive CRC cell lines, and inhibition of migration in all cell lines tested. Decreased phospho-paxillin levels may be responsible for the pronounced inhibition of migration observed after dual treatment with dasatinib and CNTO 95 in these CRC cell lines. These data support the rationale for combined Src/integrin inhibition in colon cancer, and further suggest approaches to patient selection strategies. [Table: see text]

Activity of dasatinib with chemotherapy in triple-negative breast cancer cells

e14605 Background: Triple-negative breast cancers (TNBC) lack expression of oestrogen, progesterone, and are HER-2 normal. TNBC cell lines have displayed greater sensitivity to growth inhibition by the multi-target kinase inhibitor, dasatinib, than luminal or HER- 2 positive breast cancer cell lines. The aim of this study was to assess the direct anti-tumor effects of dasatinib in combination with chemotherapy in TNBC. Methods: Four TNBC cell lines (MDA-MB-231, HCC-1143, HCC-1937, MDA-MB-468) were treated with dasatinib in combination with docetaxel, cisplatin or 5'-5' DFUR. IC50 values were calculated for each drug alone by determining response in a 5-day proliferation (acid phosphatase) assay. Combination index (CI) values were determined, using CalcuSyn, to assess the interaction between drugs. Results: Three of the cell lines (MDA-MB-231, HCC- 1143, HCC-1937) were sensitive to dasatinib (IC50 &lt; 1 μM) whereas MDA-MB-468 was resistant (IC50 &gt; 1 μM) (Table). In MDA-MB-231 and HCC-1143 cells, combined treatment with dasatinib and 5'-5'-DFUR displayed synergy (CI&lt;1.0), whereas the combination was additive in HCC-1937 cells (CI=0.98). Combined treatment with dasatinib and cisplatin was synergistic in the three dasatinib sensitive cell lines (CI&lt;1.0). Dasatinib in combination with docetaxel displayed moderate synergy in MDA-MB-231 and HCC-1937 cells (CI&lt;1.0), but was antagonistic in HCC-1143 cells (CI&gt;1.0). Conclusions: Our findings show that the combination of dasatinib with either 5'-5'-DFUR or cisplatin is synergistic in TNBC cell lines, and suggest that combinations of dasatinib with chemotherapy may improve response in triple negative breast cancer patients. [Table: see text] No significant financial relationships to disclose.