Quanhua Xing

Identification of a Natural Compound by Cell-Based Screening That Enhances Interferon Regulatory Factor-1 Activity and Causes Tumor Suppression

The transcription factor interferon regulatory factor-1 (IRF-1) is induced by many tumor-suppressive stimuli and can mediate antiproliferative and proapoptotic effects in cancer cells. Thus, identifying agents that enhance IRF-1 activity may be an effective approach to cancer therapy. A cell-based screening assay was developed to identify extracts and compounds that could enhance IRF-1 activity, using an IRF-1–dependent luciferase reporter cell line. Through this approach, we identified a natural product extract and a known active component of this extract, baicalein, which causes a marked increase in IRF-1–dependent reporter gene expression and IRF-1 protein, with modulation of known IRF-1 targets PUMA and cyclin D1. Baicalein causes suppression of growth in vitro in multiple cancer cell lines in the low micromolar range. IRF-1 plays a role in this growth suppression as shown by significant resistance to growth suppression in a breast cancer cell line stably transfected with short hairpin RNA against IRF-1. Finally, intraperitoneal administration of baicalein by repeated injection causes inhibition of growth in both xenogeneic and syngeneic mouse models of cancer without toxicity to the animals. These findings indicate that identifying enhancers of IRF-1 activity may have utility in anticancer therapies and that cell-based screening for activation of transcription factors can be a useful approach for drug discovery. Mol Cancer Ther; 10(10); 1774–83. ©2011 AACR.

Effect of eribulin on cell growth and PI3K pathway activity with and without RAD001 in triple-negative and HER2-expressing breast cancer.

173 Background: Patients with triple-negative breast cancer have high levels of Akt expression and activation of the PI3K-mTOR pathway. Eribulin is a microtubule-targeting agent with benefits in treating refractory triple negative disease. Our objective was to evaluate its efficacy in inhibiting PI3K pathway activity and cell growth both alone and in combination with the mTOR inhibitor RAD001. METHODS MDA468, BT549 and SKBR3 breast cancer cell lines were used for this study. MTT assays were used to assess growth inhibition after 72 hour treatment with eribulin alone and in combination with RAD001. Combination indices (CI) generated by Chou-Talalay plots were used to quantify synergy. Western blots were used to evaluate the expression of phosphorylated Akt-Ser473 (pAkt) and S6K1 after 24 hours of treatment with both agents. RESULTS Both MDA468 and SKBR3 cells treated with eribulin in varying concentrations showed inhibition of pAkt expression. Standard dilutions of eribulin in combination with log dilutions of RAD001 resulted in marked synergistic growth inhibition (CI<<1) in both MDA468 and BT549 cells. Western blot analysis for MDA468 cells treated with the combination erubulin and RAD001 showed a dose related suppression of pAkt along with complete inhibition of pS6K1, while RAD001 alone increased pAkt. CONCLUSIONS Our study shows dose related inhibition of Akt activation as well as inhibition of cell growth in triple negative breast cancer and HER2 cell lines treated with eribulin alone or combined with RAD001. We also show reversal of the pAkt feedback response seen with mTOR inactivation, and a significant synergistic growth inhibition with combination treatment. These findings point to a potential role for eribulin and RAD001 in the treatment of refractory triple-negative breast cancer. [Table: see text].

Abstract P3-03-15: Synergistic suppression of triple negative breast cancer with the combination of PI3K inhibitor (alpelisib, BYL719) and CDK inhibitor (ribociclib, LEE011)

INTRODUCTION. Triple negative breast cancer (TNBC) is an aggressive form of BC that lacks effective targeted therapy. It is a biologically heterogeneous disease with several molecular subtypes: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal-stem-like (MSL), immune-modulatory (IM) and unclassified (UNC). The PI3K/AKT/mTOR pathway affects cell proliferation, survival, and apoptosis through growth receptor interaction with downstream targets such as AKT and mTOR. TNBC frequently has activation of this pathway by mutation and other means across several subtypes. Recently the PI3K inhibitor alpelisib (BYL719) has been found to have efficacy in a combination study in ER positive metastatic BC (MBC) with a Phase 3 trial in progress. Ribociclib (LEE011) is a CDK inhibitor targeting cyclin D1/CDK4 and cyclin D3/CDK6 that recently met its primary endpoint in a phase 3 trial in a combination study in hormone receptor positive BC. As with all BC, TNBC has intact or mutant RB which affects susceptibility to CDK inhibitor. We hypothesize that targeting both PI3K with alpelisib and CDK with ribociclib may provide enhanced suppression of TNBC. MATERIALS AND METHODS. HCC38 and MDA468 are TNBC subtype BL1; MDA468 is RB1 and PTEN mutant. HCC1187 is IM. BT549 is M, mutant RB1 and PTEN. Hs578T, MDA157, and MDA231 are MSL. Cells were treated with alpelisib and ribociclib alone with 2-fold increase in concentrations, or the combination, for 72h and assessed by MTT assay. Western blot was performed using probes for pAkt (T308 and S473), Akt, pS6K1, S6K1, pS6, S6, and β-actin. RESULTS. Synergy in growth inhibition was seen combining alpelisib and ribociclib as reflected by combination indices Conclusion. Our study demonstrates that alpelisib plus ribociclib can synergistically enhance suppression of BC across multiple subtypes of TNBC. Cancer cells within the same subtype may not demonstrate a synergistic response, depending on RB status. Nevertheless, if RB mutant cells are susceptible to ribociclib then synergy can be seen even in these cancer cells. Antagonism can be seen independent of RB status in the same subtype. These findings point to a potential role for combination therapy with alpelisib and ribociclib in the treatment of TNBC. Citation Format: Yuan Y, Mortimer J, Xing Q, Yan J, Wen W, Han E, Yim JH. Synergistic suppression of triple negative breast cancer with the combination of PI3K inhibitor (alpelisib, BYL719) and CDK inhibitor (ribociclib, LEE011) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-15.

Abstract 2183: Baicalein and meformin decrease small cell lung cancer growth by inhibiting the mTOR pathway in itro

Background: Metformin use has been associated with decreased lung cancer risk in several observational studies and is currently in clinical trial in conjunction with standard chemotherapeutics. One possible antitumor mechanism is the negative regulation of the mTOR pathway. We previously reported that the natural product Baicalein has antitumor effects through targeting mTOR pathway. We hypothesized that Baicalein to have antitumor effects in small cell lung cancer. Methods: H1417 small cell lung cancer cells were cultured and treated with increasing doses of Baicalein or Metformin. The cells were harvested at 24 hours and subjected to Western blotting staining for the downstream products of the mTOR pathway. Cell proliferation was determined by MTT assay at 24, 48, and 72 hours. MTT conversion to formazan dye correlates with the number of living cells. Results: We found a dose dependent decrease in the downstream mTOR1 targets pS6K1 and pS6 using both Baicalein and Metformin. There was also an increase in the expression of the mTOR inhibitors DDIT4 and IRF-1. Using the MTT assay, we were able to demonstrate a marked dose dependent decrease in cell proliferation that was sustained over 72 hours from treatment. Interestingly, Baicalein has a markedly higher potency working at micromolar doses verses Metformin which required millimolar doses. Conclusions: Both Baicalein and Metformin effectively decrease cell proliferation in small cell lung cancer cells in vitro. We have shown these drugs to target the mTOR pathway. Cell proliferation is inhibited at a markedly smaller dose by Baicalein compared with Metformin. Baicalein may be useful in cancer chemotherapy and chemoprevention. Citation Format: Emily F. Marcinkowski, Dan Raz, Bing Shen, Quanhua Xing, Jin Yan, Wei Wen, Ernest Han, John Yim. Baicalein and meformin decrease small cell lung cancer growth by inhibiting the mTOR pathway in itro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2183.

Abstract P3-03-07: Combination of eribulin and PI3K inhibitors in triple negative and HER2 expressing breast cancer cell lines results in synergistic growth inhibition and enhanced inhibition of the PI3K pathway

Background. Patients with triple negative breast cancer (TNBC) have high levels of pAkt expression and activation of the PI3K-mTOR pathway. Eribulin is a microtubule-targeting agent with benefits in treating taxane and anthracycline refractory breast cancer. Our objective was to evaluate its efficacy in inhibiting PI3K pathway activity and cell growth both alone and in combination with PI3K/MTOR inhibitors BEZ 235 and BKM 120. Methods. TNBC cell lines MDA468, BT549, HS587T, MDA231, and HER2 expressing breast cancer cell line SKBR3 were used for this study. Both MDA468 and BT549 have PI3K- related mutations. The tetrazolium salt, 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assays were used to assess growth inhibition after 72 hour treatment with eribulin, BEZ 235 and BKM 120 both alone and in combination. Combination indices (CI) generated by Chou-Talalay plots were used to quantify synergy. Western blots were used to evaluate the expression of phosphorylated Akt (pAkt), S6K1 (pS6K1) and S6 (pS6) from 30 min to 24 hours of treatment at different doses. Results. Eribulin has IC50 ranging from 60 pM to 300 pM, BEZ 235 has IC50 ranging from 50 nM to 80 nM, and BKM has IC50 ranging from 500 nM or higher. Standard dilutions of eribulin in combination with BEZ 235 resulted in synergistic growth inhibition (CI<1) in both MDA468 and BT549 cells at all doses tested, but required higher concentrations (500 to 2000 nM) for BKM 120. Western blot analysis for all cell lines treated with eribulin showed pAkt inhibition by eribulin alone with doses as low as 1 nM and as early as 4 hours. PI3K inhibitor alone confirmed inhibition of pAkt, pS6K1 and pS6 at early time points with feedback increase in pAkt at 24h. While both BEZ 235 and BKM 120 treatment increased pAkt in a dose dependent fashion at 24 hours, combination treatment with eribulin showed a dose dependent decrease in pAkt. Conclusion . Our study shows significant synergistic growth inhibition with the combination of eribulin and PI3K inhibitors. This may be related to inhibition by eribulin of the feedback increase in pAkt seen with PI3K inhibitors alone at later time points. These findings point to a potential role for combination therapy of both eribulin and PI3K inhibitors in treating refractory metastatic disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-07.

Abstract P6-13-17: The combination of eribulin and everolimus results in enhanced suppression of tumors in mouse models of triple negative breast cancer

INTRODUCTION. Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with poor overall and relapse free survival. TNBC does not have targeted or matched therapies. Patients have worse outcomes after chemotherapy than with other subtypes of breast cancer. TNBC accounts for 12-17% of all breast cancers, leaving an unmet need for targeted therapy. Efforts to profile these tumors have revealed several potential targets. The PI3K/AKT/mTOR pathway is a signal transduction pathway that links growth related hormone receptor interaction to downstream targets such as AKT and mammalian target of rapamycin (mTOR). This pathway targets affect cell proliferation, survival, and apoptosis. Patients with TNBC have high levels of AKT expression and activation of this pathway. Microtubule-targeting agents have been used in TNBC. Eribulin mesylate is a microtubule-targeting agent with benefits in treating taxane and anthracycline refractory breast cancer via a microtubule targeting anti-mitotic mechanism. It has been approved for the treatment of TNBC in heavily pretreated patients. Despite targeted therapy, breast cancer cells can grow resistant. Targeting multiple cancer growth pathways has been used in patients that progress on therapy or fail to respond. We hypothesized that targeting both mitotic blockade and PI3K/AKT/mTOR pathway may provide enhanced suppression of TNBC growth in both syngeneic and xenogeneic mouse models. MATERIALS AND METHODS. MDA-MB-468 is a human TNBC cell line. 4T1 is a highly metastatic mouse TNBC cell line derived from a spontaneously arising Balb/c mammary tumor. 4T1 and MDA-MB-468 tumor cells were injected into the mammary fat pad of female Balb/c and NOD/SCID/IL2Rgamma null (NSG) mice (with matrigel) respectively. After tumors were formed Balb/c mice were treated three times per week with vehicle, eribulin (0.75 mg/kg i.v.), RAD001 (5 mg/kg via oral gavage) or a combination of both. NSG mice were treated three times per week with vehicle, eribulin (0.5 mg/kg i.v.), RAD001 (5 mg/kg by oral gavage), or a combination of both. Tumor volumes and body weights were measured. Student t-test was used to compare the means of two groups and determine the p value (p RESULTS. In the 4T1 syngeneic breast cancer mouse model, the combination of Eribulin and Everolimus resulted in marked suppression of tumor growth which was statistically significant versus vehicle treatment alone, or Eribulin or Everolimus alone (Table I). In the MDA-MB-468 model, the combination of Eribulin and Everolimus demonstrated marked suppression of tumor growth which was statistically significant compared to either agent alone (Table II). Citation Format: Marcinkowski E, Luu T, Yuan Y, Mortimer J, Leong L, Portnow J, Xing Q, Wen W, Yim J. The combination of eribulin and everolimus results in enhanced suppression of tumors in mouse models of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-17.

Abstract 5550: The flavone baicalein increases DDIT4 protein expression and inhibits mTOR activity in platinum-resistant epithelial ovarian cancer cells.

OBJECTIVES: Baicalein is a natural flavone and an active component in traditional Chinese medicine. We have recently found that epithelial ovarian cancer cells treated with baicalein increases DDIT4 gene expression, a mediator of DNA damage response. Our objectives are to compare the effects of baicalein to carboplatin and gemcitabine on platinum sensitive and resistant ovarian cancer cell lines. Specifically we examined DDIT4 protein expression, effect on mTOR activity, and growth inhibition. METHODS: The ovarian cancer cell line A2780 and the derivative platinum resistant cell line A2780R were treated with carboplatin, gemcitabine, and baicalein with appropriate controls and growth was assessed at 72 hours by MTT assay. Alternatively cells were treated at identical concentrations for 24 hours and western blots were performed for DDIT4 protein expression, mTOR activity as reflected by phosphorylation of S6K1 (pS6K1) and S6 (pS6), as well as unphosphorylated controls and actin. RESULTS: MTT assay demonstrated that carboplatin and gemcitabine do not inhibit the growth of the A2780R cells at concentrations that inhibit A2780 while baicalein inhibits both cell lines nearly equally (2.5 to 20 μM). DDIT4 protein expression increased in a dose dependent fashion in both cell lines treated with baicalein. Furthermore, the mTOR targets S6K1 and S6 demonstrate a dose dependent decrease in phosphorylation, reflecting inhibition of mTOR activity (Figure 1). In contrast, DDIT4 protein expression was not altered in carboplatin and gemcitabine treated A2780R cells with minimal effect on mTOR activity, whereas A2780 cells displayed increased DDIT4 protein with increasing doses as well as corresponding decreases in the phosphorylation of the mTOR targets. CONCLUSIONS: Baicalein can inhibit growth of both platinum sensitive and resistant ovarian cancer cells. The inhibitory effects of baicalein correlate with both DDIT4 protein expression and inhibition of mTOR activity as reflected by phosphorylation of the mTOR targets S6K1 and S6. Baicalein may be a novel treatment option for patients with ovarian cancer, especially those that are platinum resistant. DDIT4 may mediate growth inhibition through inhibition of mTOR. Interestingly, DDIT4 expression and inhibition of mTOR activity may also play a role in the antitumor effects of the chemotherapeutics carboplatin and gemcitabine in sensitive ovarian cancer cells. Citation Format: Ernest S. Han, Yujun Wang, Quanhua Xing, Jin Yan, Richard Yip, Mark Wakabayashi, John Yim. The flavone baicalein increases DDIT4 protein expression and inhibits mTOR activity in platinum-resistant epithelial ovarian cancer cells. [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 5550. doi:10.1158/1538-7445.AM2013-5550

Abstract P3-03-08: A comparison of PI3K inhibition by eribulin, other microtubule targeting agents and a DNA-damaging chemotherapeutic in triple negative and HER2 expressing breast cancer cell lines

Background. Eribulin is a microtubule-targeting agent with significant benefits in treating refractory metastatic breast cancer. Tumors from patients with Triple negative breast cancer (TNBC) have high levels of Akt expression and consistently show activation of the PI3K-mTOR pathway. Our objective was to compare Eribulin9s ability to inhibit PI3K pathway activity and cell growth with two other microtubule targeting agents, Paclitaxel and Vinblastine, as well as a conventional DNA damaging chemotherapeutic Cisplatin. Methods. MDA468 and BT549 TNBC cell lines and SKBR3 HER2 overexpressing breast cancer cell lines were used for this study. Western blot analysis was used to evaluate the expression of phosphorylated Akt-Ser473 (pAkt) and S6K1 (pS6K1) at different time points from 2 to 24 hours of treatment with Eribulin, Paclitaxel, Vinblastine, or Cisplatin. MTT assays were used to assess growth inhibition after 72 hours of treatment. Results. Western blot analysis for MDA468 cells treated with Eribulin in varying concentrations confirm partial inhibition of pAkt expression as early as 4 hours at 100 pM concentration. Complete inhibition is reached at 50 nM. Partial inhibition of pS6K1 can be seen as early as 4 hours at 500 nM. Western analysis for MDA468 cells treated with Vinblastine in varying concentrations confirms inhibition of pAkt and pS6K1 beginning at 50 nM at 24 hours. Western analysis for MDA468 cells treated with Paclitaxel in varying concentrations showed increases in pAkt expression in a dose responsive fashion with significant increase in pAkt beginning at 5 nM concentration as well as increase in pS6K1 at 24 hours. Cisplatin markedly increases pAkt at 24 hours in a dose responsive fashion and decreases pS6K1 at 500 nM to 1000 nM concentration range in BT549. The IC509s for Eribulin ranged from 0.06 nM to 0.3 nM at 72 hours by MTT assay. The IC509s for Vinblastine ranged from 0.5 nM to 0.9 nM. Paclitaxel has reported IC509s in the 2 nM to 75 nM range in these cell lines, and Cisplatin has IC509s ranging from to ∼500 nM to ∼2000 nM at 72 hours in these cell lines. Conclusion . Our study shows that for microtubule targeting agents such as Eribulin and Vinblastine that block polymerization of tubulin into microtubules, both pAkt and pS6K1 expression is suppressed. Growth inhibition is also confirmed, and is seen at doses when pAkt and pS6K1 are not suppressed. Eribulin inhibits pAkt and pS6K1 at lower concentrations than Vinblastine. With Paclitaxel, a microtubule-targeting agent that enhances polymerization of tubulin to microtubules, and Cisplatin, a conventional DNA damaging chemotherapeutic we observe an increase in pAkt expression, with variable effects on pS6K1. Enhancement of Akt activity is a likely survival response by cancer cells to chemotherapy, yet in the case of microtubule polymerization blockade as seen with Eribulin and Vinblastine, Akt activity is suppressed, along with downstream mTOR activity. The potential role of microtubule polymerization blockade in inhibition of the PI3K pathway needs further study. It may be a factor in the novel finding of pAkt and pS6K1 inhibition by Eribulin. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-08.

Abstract P6-14-13: A Flavone from Traditional East Asian Natural Extract Inhibits STAT3 and Tumor Growth in a Syngeneic Mouse Breast Cancer Model

Background: Recent studies have shown that increased dietary consumption of flavones and isoflavones is associated with reduced risk of developing cancer. Natural plants/herbs used in Traditional East Asian Medicine (TEAM) have been recognized as potentially efficacious natural treatments against cancer and are enriched with isoflavones and flavones, as well as other interesting compounds. We used a novel high throughput screening method to identify natural extracts that may have a potential anti-cancer effect and identified the TEAM natural product Scutellaria baicalensis and a known active component, the flavone, baicalein. The objective of our current study is to investigate the anti-cancer effects of baicalein in vitro and in vivo. Methods: In vitro testing was performed in mouse (C3L5) and human (SKBR3) breast cancer cell lines. Cell viability was assessed by MTT assay. Apoptosis was assessed using FITC-Annexin V and PI staining with bivariate reading by flow cytometry at 48 h. A syngeneic mouse breast cancer model (C3H/HeJ — C3L5) was used for in vivo testing. Mice bearing subcutaneous tumors were either treated with daily intraperitoneal injection of vehicle or baicalein (20 mg/kg) five days a week for three weeks (n=6/group). Tumor size and weights were measured. Results: Baicalein had a marked cytotoxic effect in C3L5 cells with an IC50 of less than 10mM (data not shown). FITC-Annexin V and PI analysis with flow cytometry for C3L5 cells treated at 10μM for 48h showed a marked increase in the Annexin V positive fraction indicative of apoptosis. In vivo studies demonstrated a significantly decreased tumor growth in mice treated with intraperitoneal baicalein when compared to the control group (P Baicalein treated cells demonstrated STAT3 inhibition on western blot analysis by decreased presence of phospho-STAT3 (data not shown). Conclusions: Baicalein, a flavone from TEAM demonstrates significant anti-cancer effects due to apoptosis in breast cancer. STAT3, a well-known anti-apoptotic signaling intermediate, is inhibited in activation as reflected by decreased phosphoSTAT3 which may account for these effects. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-13.