Hyun-Jin Nam

OPB-31121, a novel small molecular inhibitor, disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells

We investigated the mechanisms of action and antitumor effects of OPB-31121, a novel STAT3 inhibitor, in gastric cancer cells. OPB-31121 downregulated JAK2 and gp130 expression and inhibited JAK2 phosphorylation which leads to inhibition of STAT3 phosphorylation. OPB-31121 inhibited constitutively activated and IL-6-induced JAK/STAT signaling pathway. OPB-31121 decreased cell proliferation in both gastric cancer cells and in a xenograft model, induced the apoptosis of gastric cancer cells, inhibited the expression of antiapoptotic proteins, and showed synergism with 5-fluorouracil and cisplatin. Taken together, our study suggests that STAT3 inhibition with OPB-31121 can be tested in patients with gastric cancer.

Evaluation of the antitumor effects and mechanisms of PF00299804, a pan-HER inhibitor, alone or in combination with chemotherapy or targeted agents in gastric cancer

Recently, HER2-directed treatment, such as trastuzumab, has shown clinical benefit in HER2-amplified gastric cancer. On the basis of recent studies about epidermal growth factor receptor (EGFR) or HER2-targeting agents (including gefitinib, lapatinib, and trastuzumab) in gastric cancer, the potent effects of pan-HER inhibitors targeting the HER family are anticipated. In this study, we evaluated the activity and mechanisms of PF00299804, an irreversible pan-HER inhibitor, in gastric cancer in vitro and in vivo models. PF00299804 showed significant growth-inhibitory effects in HER2-amplified gastric cancer cells (SNU216, N87), and it had lower 50% inhibitory concentration values compared with other EGFR tyrosine kinase inhibitors, including gefitinib, lapatinib, BIBW-2992, and CI-1033. PF00299804 induced apoptosis and G1 arrest and inhibited phosphorylation of receptors in the HER family and downstream signaling pathways including STAT3, AKT, and extracellular signal–regulated kinases (ERK) in HER2-amplified gastric cancer cells. PF00299804 also blocked EGFR/HER2, HER2/HER3, and HER3/HER4 heterodimer formation as well as the association of HER3 with p85α in SNU216 cells. The combination of PF00299804 with clinically relevant chemotherapeutic agents or molecular-targeted agents including trastuzumab (an anti-HER2 monoclonal antibody), CP751871 (an IGF1R inhibitor), PD0325901 (an ERK1/2 inhibitor), and PF04691502 (a PI3K/mTOR inhibitor) produced synergistic effects. These findings indicate that PF00299804 can be used as a targeted therapy for the treatment of HER2-amplified gastric cancer through inhibition of HER family heterodimer formation and may augment antitumor efficacy of chemotherapeutic and/or molecular-targeted agents. Mol Cancer Ther; 11(2); 439–51. ©2011 AACR.

RAD51C-Deficient Cancer Cells Are Highly Sensitive to the PARP Inhibitor Olaparib

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

Antitumor Activity of Saracatinib (AZD0530), a c-Src/Abl Kinase Inhibitor, Alone or in Combination with Chemotherapeutic Agents in Gastric Cancer

Src is a nonreceptor tyrosine kinase involved in the cross-talk and mediation of many signaling pathways that promote cell proliferation, adhesion, invasion, migration, and tumorigenesis. Increased Src activity has been reported in many types of human cancer, including gastric cancer. Therefore, this factor has been identified as a promising therapeutic target for cancer treatments, and targeting Src in gastric cancer is predicted to have potent effects. We evaluated the antitumor effect of a c-Src/Abl kinase inhibitor, saracatinib (AZD0530), alone or combined with chemotherapeutic agents in gastric cancer cell lines and a NCI-N87 xenograft model. Among 10 gastric cancer cell lines, saracatinib specifically inhibited the growth and migration/invasion of SNU216 and NCI-N87 cells. Saracatinib blocked the Src/FAK, HER family, and oncogenic signaling pathways, and it induced G1 arrest and apoptosis in SNU216 and NCI-N87 cells. Apoptosis required induction of the proapoptotic BCL2 family member Bim. Knockdown of Bim using siRNA decreased apoptosis induced by treatment with saracatinib, suggesting that Bim has an important role in saracatinib-induced apoptosis. Saracatinib enhanced the effects of lapatinib, an EGFR/HER2 dual inhibitor, in SNU216 and NCI-N87 cells. Furthermore, combined treatment with saracatinib and 5-fluorouracil (5-FU) or cisplatin exerted synergistic effects in both saracatinib-sensitive and saracatinib-resistant cells. Consistent with our in vitro findings, cotreatment with saracatinib and 5-FU resulted in enhanced antitumor activity in the NCI-N87 xenografts. These data indicate that the inhibition of Src kinase activity by saracatinib alone or in combination with other agents can be a strategy to target gastric cancer. Mol Cancer Ther; 12(1); 16–26. ©2013 AACR.

Antitumor activity of HM781-36B, an irreversible Pan-HER inhibitor, alone or in combination with cytotoxic chemotherapeutic agents in gastric cancer

Trastuzumab, a HER2 directed treatment has shown clinical benefit in HER2 amplified gastric cancer. This study demonstrated the potent antitumor activity of HM781-36B, a quinazoline-based irreversible pan-HER inhibitor, in HER2 amplified gastric cancer cells (SNU216 and N87) in vitro and in vivo. HM781-36B inhibited phosphorylation of HER family and downstream signaling molecules, and induced apoptosis and G1 arrest. Furthermore, HM781-36B exerted synergistic effects with chemotherapeutic agents in both HER2 amplified and HER2 non-amplified gastric cancer cells. Therefore, HM781-36B may be useful for the treatment of HER2 amplified gastric cancer alone or in combination with chemotherapeutic agents.

The irreversible pan-HER inhibitor PF00299804 alone or combined with gemcitabine has an antitumor effect in biliary tract cancer cell lines

SummaryBiliary tract cancer (BTC) is associated with poor survival and unresponsiveness to chemotherapy. Targeted therapies for BTC have been studied, and HER family members are promising therapeutic targets in BTC. In this study, we evaluated the efficacy of PF00299804, an irreversible pan-HER inhibitor, in eight BTC cell lines alone or combined with gemcitabine. PF00299804 potently inhibited the growth of two cell lines (SNU308 and SNU478) out of the eight BTC cell lines as a single agent. PF00299804 blocked HER family and downstream signaling pathways, inducing G1 arrest or apoptosis. Moreover, PF00299804 exerted synergistic effects with gemcitabine in seven of the eight BTC cell lines, possibly through the regulation of the genes involved in the response to gemcitabine, such as TS (thymidylate synthase), RRM1 (ribonucleotide reductase), and MAGEH1, which is negatively correlated with gemcitabine sensitivity. Our results support the need for further study of PF00299804 alone or combined with gemcitabine for the treatment of BTC.

Anti-tumor Effect of KX-01 Through Inhibiting Src Family Kinases and Mitosis

Purpose KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. Materials and Methods The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. Results KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. Conclusion KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Abstract 747: Evaluation of Src as a therapeutic target and development of biomarkers of Src inhibitor in cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, invasion and angiogenesis. Elevation of Src activity has been reported in many types of cancers including gastric cancer (GC) and biliary tract cancer (BTC). The purpose of this study is to evaluate Src as a therapeutic target and to elaborate the biomarkers of Src inhibitor in GC and BTC. Methods: Ten gastric cancer cell lines (SNU-1, 5, 16, 216, 601, 620, 638, 668, 719, NCI-N87) and 8 biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, TFK1) were used. Saracatinib and bosutinib were used as Src inhibitors. MTT assay, colony formation assay and 3D culture were done for determining growth inhibitory effect of Src inhibitors, alone or in combination with chemotherapeutic agents (5-FU, gemcitabine, cisplatin). Cell cycle analysis was done by FACS Calibur flow cytometer. Matrigel invasion assay and wound healing assay were done. The methods described by Chou and Talalay were used to determine whether a synergistic effect existed between drug combination. Tumor xenograft model was made and used for in vivo test of Src inhibitors. Results: Among 10 GC cells, SNU216 and NCI-N87 were sensitive to Src inhibitor. These sensitive cells showed high levels of pSRC(Y416) and pFAK (Y861, Y397, Y925). These 2 sensitive GC cells are both HER2 amplified cells. However, HER2-positive breast cancer (BC) cells (SKBR3, BT474, MDA-MB453) were resistant to Src inhibitor. Contrast to these resistant BC cells, SNU216 and N87 showed high expression of integrin αV, β4 and β8. Especially, in case of integrin β8, the mRNA/protein levels were highest in SNU216 and N87 among all GC cells. Src inhibitor-sensitive GC cells showed the apoptosis by Src inhibitor and synergism with 5-FU in vitro and in vivo. Among 8 BTC cells, 3 cells were sensitive to Src inhibitor (SNU308, SNU478 and HuCCT1) in terms of growth inhibition and migration/invasion inhibition. Sensitive cells showed high levels of integrin α2, α3 and β4. Src inhibitor induced G1 arrest and decreased pSrc, pFAK, and pERK in sensitive cells. Inhibition of pSrc was accompanied with increase of PTEN and decrease of pAKT. Src inhibitor showed the synergistic effects with cytotoxic chemotherapeutic agents (gemcitabine and cisplatin) in vitro and in vivo. When the Src was inhibited, pSTAT3 was increased thru increase of IL-6 in some cells. Conclusion: Taken together, Src could be a potential therapeutic target in gastric cancer and biliary tract cancer. The role of integrin as a biomarker for Src inhibitor should be further investigated. Citation Format: Ah-Rong Nam, Hyun-Jin Nam, Kyo Hwa Kang, Ji Eun Park, Tae Yong Kim, Sae-Won Han, Sang-Hyun Song, Seock-Ah Im, Tae-You Kim, Do-Youn Oh, Yung-Jue Bang. Evaluation of Src as a therapeutic target and development of biomarkers of Src inhibitor in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 747. doi:10.1158/1538-7445.AM2014-747

Abstract 2386: Amphiregulin confers trastuzumab resistance by activating PI3K/Akt pathway in HER2-positive breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Amphiregulin is a ligand for the epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor 2 (HER2) shares common signal pathways and forms a heterodimer with EGFR. In this study, we investigated the effect of amphiregulin on trastuzumab therapy in HER2-positive breast cancer. Methods: We analyzed serum amphiregulin levels by enzyme-linked immunosorbent assay (ELISA) from baseline serum samples obtained from HER2-positive metastatic breast cancer patients who received first-line trastuzumab plus taxane chemotherapy. In addition, in vitro experiments were performed to elucidate the biologic mechanism of clinical findings related to amphiregulin using SK-BR-3 and BT-474 cell lines. Results: Between October 2004 and July 2009, a total of 50 women with HER2-positive metastatic breast cancer were consecutively enrolled. The median age was 47 years (range, 27-72 years). Eighteen patients (36.0%) received weekly paclitaxel plus trastuzumab, 24 patients (48.0%) tri-weekly paclitaxel plus trastuzumab, and 8 patients (16.0%) tri-weekly docetaxel plus trastuzumab. Among 43 patients with measurable lesions, the response rate (RR) was 76.7%. The median follow-up duration was 29.2 months (range, 0.7-63.3 months). The median progression-free survival (PFS) was 17.6 months (95% confidence interval (CI), 13.4-21.9 months). The median overall survival (OS) was 47.0 months (95% CI, 35.3-58.6 months). The median serum amphiregulin level was 1.0 ng/mL with a maximum level of 4.4 ng/mL. Patients with high serum amphiregulin levels (≥0.5 ng/mL) had significantly shorter PFS (p=0.018) along with a tendency toward lower RR (p=0.237) and shorter OS (p=0.529) than the others. The in vitro colony forming assay demonstrated that the addition of amphiregulin resulted in increased proliferation of both SK-BR-3 and BT-474 cells. In addition, the anti-proliferative effect of trastuzumab was decreased in the presence of amphiregulin in both SK-BR-3 and BT-474 cells. The Western blot analysis showed that amphiregulin increased the phosphorylation of Akt and its downstream molecules in both SK-BR-3 and BT-474 cells. In addition, in the presence of amphiregulin, sustained phosphorylation of Akt and its downstream molecules was observed after trastuzumab treatment in both SK-BR-3 and BT-474 cells. Conclusions: High serum amphiregulin levels (≥0.5 ng/mL) predicted disease progression after first-line trastuzumab plus taxane chemotherapy in patients with HER2-positive metastatic breast cancer. Amphiregulin promoted the proliferation of HER2-positive breast cancer cells in vitro and induced trastuzumab resistance by activating PI3K/Akt pathway. Our results suggest that the measurement of serum amphiregulin levels by ELISA may provide additional information for the clinical outcome of trastuzumab-based chemotherapy in patients with HER2-positive breast cancer. Citation Format: Ji-Won Kim, Young Seok Joung, Ahrum Min, Hyun-Jin Nam, Jee Hyun Kim, Seock-Ah Im, Kyung-Hun Lee, Jin-Soo Kim, Tae-Yong Kim, Sae-Won Han, Yoon Kyung Jeon, Do-Youn Oh, Tae-You Kim, In Ae Park. Amphiregulin confers trastuzumab resistance by activating PI3K/Akt pathway in HER2-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2013-2386

Abstract 3588: Antitumor activity of saracatinib, a c-Src/Abl kinase inhibitor, in gastric cancer

Background: Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, adhesion, invasion, migration, metastasis, angiogenesis, and tumorigenesis. Elevation of Src activity has been reported in many types of human cancers. Therefore, Src has been promising therapeutic target for the treatment of cancers. However, the mechanism of Src inhibition in gastric cancers has not been fully understood yet. Saracatinib is an orally active small molecule c-Src/abl kinase inhibitor currently in phase II clinical trials. The purpose of this study is to evaluate the therapeutic potential of saracatinib alone or in combination with chemotherapeutic agents for the treatment of gastric cancers by testing its in vitro and in vivo efficacy. Methods: Experiments were conducted in human gastric cancer cell lines from the Korea Cell Line Bank or the American Type Culture Collection. MTT assay was used for determining growth inhibitory effect of saracatinib alone or in combination with chemotherapeutic agents (5-FU, cisplatin). Cell cycle distribution was analyzed by FACS Calibur flow cytometer and apoptotic effect was measured by annexin V assay. Western blot analysis was performed to verify molecular mechanism of this compound. The methods described by Chou and Talalay were used to determine if a synergistic effect existed. The analysis of the median effect was conducted using the Calcusyn software to determine a combination index value. (CI>1: antagonistic effect, CI=1:additive effect, CI Results: Saracatinib specifically inhibited the growth of SNU216 and NCI N87 cells among 10 gastric cancer cell lines. We found that saracatinib induces G1 arrest and apoptosis in sensitive cell lines. The apoptotic process required induction of the proapoptotic BH3-only BCL2 family member, BIM, and saracatinib led to increase in the BIM level in SNU216 and N87 cells. Knockdown of BIM using siRNA decreased the apoptotic cell numbers induced by treatment of saracatinib suggesting important role for BIM in saracatinib induced apoptosis pathway. The sensitivity to saracatinib in SNU216 and N87 cell lines was correlated with inhibiton of AKT and ERK pathways and blockade of HER family kinases. Furthermore, the combined treatment of saracatinib with 5-FU or cisplatin exerted synergistic effects in gastric cancer cell lines including NCI N87 cells. Consistent with these in vitro findings, saracatinib alone or in combination with 5-FU showed antitumor activity in a NCI-N87 xenograft model. Conclusion: Taken together, these preclinical evaluations support that Src is a potential therapeutic target in gastric cancer and clinical development of saracatinib alone or in combination with chemotherapy in gastric cancer could be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3588. doi:10.1158/1538-7445.AM2011-3588