Paclitaxel In Breast Cancer Research Articles

Acquisition of epithelial–mesenchymal transition is associated with Skp2 expression in paclitaxel-resistant breast cancer cells

Background:Breast cancer is the most common female malignant disease, and the second leading cause of cancer-related death in the United States. Acquired resistance to chemotherapeutic drugs is a pivotal reason that leads to worse treatment outcome of breast cancer. Therefore, it is urgent to elucidate the mechanism of drug resistance in breast cancer.Methods:To investigate the underlying molecular basis of the acquired resistant cells to paclitaxel in breast cancer, we used multiple methods including real-time RT–PCR, western blotting analysis, migration and invasion assays, wound healing assay, and transfection.Results:We found that epithelial–mesenchymal transition (EMT) is involved in paclitaxel-resistant (PR) breast cancer cells. The resistant cells with EMT features exhibit increased migration and invasion activities. Mechanistically, high expression of Skp2 was found to be associated with EMT in PR cells. Notably, depletion of Skp2 in PR cells led to partial reversal of EMT phenotype.Conclusions:These findings suggest that Skp2 was critically involved in PR-mediated EMT. Skp2 could be a potential therapeutic target for breast cancer.

Silencing MAP3K1 expression through RNA interference enhances paclitaxel-induced cell cycle arrest in human breast cancer cells

The objective of this study is to compare the expression level of MAP3K1 between normal mammary gland cells and breast cancer cells, and to analyze the effects of silencing MAP3K1 on breast cancer cells with paclitaxel treatment. Western blotting analysis was used to detect the expression level of MAP3K1 in MCF-7 and MCF-12F cells. The effect of gene silencing through different siRNAs was determined by realtime-PCR. MTT assay was used to test the cell proliferation. Cell cycle was detected by flow cytometry. MAP3K1 protein expression level in breast cancer cells was higher than that in normal mammary gland cells. MAP3K1 siRNA transfection significantly reduced the expression level of MAP3K1, and enhanced paclitaxel-induced cell proliferation inhibition and cell cycle arrest in breast cancer cells. Targeting MAP3K1 expression through small RNA interference can promote the therapeutic effects of paclitaxel in breast cancer.

TLR4 Is a Novel Determinant of the Response to Paclitaxel in Breast Cancer

Overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. We found that TLR4 is overexpressed in the majority of clinical breast cancer samples and in 68% of the examined breast cancer lines. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug paclitaxel. LPS is frequently used to show a tumor-promoting role of TLR4 although this bacterial component is unlikely to be found in the breast cancer environment. We reasoned that paclitaxel-dependent activation of TLR4 is more relevant to breast cancer chemoresistance that could be mediated by activation of the NF-κB pathway leading to upregulation of prosurvival genes. To test this hypothesis, we correlated TLR4 expression with resistance to paclitaxel in two modified breast cancer lines with either depleted or overexpressed TLR4 protein. Depletion of TLR4 in naturally overexpressing MDA-MB-231 cells downregulated prosurvival genes concomitant with 2- to 3-fold reduced IC(50) to paclitaxel in vitro and a 6-fold decrease in recurrence rate in vivo. Conversely, TLR4 overexpression in a negative cell line HCC1806 significantly increased expression of inflammatory and prosurvival genes along with a 3-fold increase of IC(50) to paclitaxel in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. Importantly, both tumor models showed that many paclitaxel-upregulated inflammatory cytokines were coinduced with their receptors suggesting that this therapy induces autocrine tumor-promoting loops. Collectively, these results show that paclitaxel not only kills tumor cells but also enhances their survival by activating TLR4 pathway. These findings suggest that blocking TLR4 could significantly improve response to paclitaxel therapy.

Is a Dexamethasone-Sparing Strategy Capable of Preventing Acute and Delayed Emesis Caused by Combined Doxorubicin and Paclitaxel for Breast Cancer? Analysis of a Phase II Trial

Objective: The effectiveness of palonosetron without delayed dexamethasone dosing against emesis was investigated in patients scheduled to receive the corticosteroid-containing combination of doxorubicin and paclitaxel (AT) for 3 cycles. Methods: Chemo-naïve women with breast cancer receiving doxorubicin (60 mg/m²) and paclitaxel (200 mg/m²) were eligible. Patients received palonosetron 0.25 mg intravenously before chemotherapy, however, all patients also received a premedication consisting of prednisone (25 mg orally the evening before therapy) and hydrocortisone (250 mg intravenously just before paclitaxel). The primary end point was complete control (CC; no vomiting, no rescue anti-emetics, and no more than mild nausea) during the overall phase (days 1-5) following cycle 1. Results: Seventy-six patients were enrolled and evaluable (median age 50 years). Fifty-six patients (74%; 95% CI 62-83%) achieved overall CC. Acute (day 1) and delayed (days 2-5) CC rates were 78 and 74%, respectively. No vomiting rates for the acute, delayed and overall phases were 85, 85 and 83%, respectively. An exploratory analysis showed only a small decrease in the probability of achieving CC between cycle 1 (74%) and cycle 3 (66%). Conclusion: The dexamethasone-sparing strategy prevented emesis in more than 70% of breast cancer patients receiving their initial cycle of AT chemotherapy.

Abstract P6-11-03: Site-Specific, Concomitant Delivery of Rapamycin and Paclitaxel in Breast Cancer: Consequent Synergistic Efficacy Enhancement

Abstract Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) (PI3k/Akt/mTOR) pathway is dysregulated in certain breast cancers. Ongoing clinical trials aim to therapeutically exploit this pathway through administration of rapamycin (RAP), an mTOR inhibitor, in combination with paclitaxel (PTX). However, actual drug synergy in clinical settings may not be fully realized due to disparate pharmacokinetic parameters of individual drug formulations, wherein drugs or their effects may never be present in the tumor at the same time. Our objective was to generate a nanoparticle platform capable of site-specifically delivering precise amounts of rapamycin and paclitaxel to breast tumors with hopes of increasing synergistic targeting of the PI3k/Akt/mTOR pathway. Materials and Methods: Drug-containing nanoparticles composed of amphiphilic block copolymers of pegylated poly(∈-caprolactone) (PEG-PCL, MW = 5k-5k) were fabricated and nanoparticle size and drug loading efficiency was determined. In vitro growth inhibition of nanoparticle formulations of varying ratios was evaluated in MCF-7 and MDA-MB-468 breast cancer cells via sulforhodamine B assays, after which median-effect plot analyses and combination index calculations were conducted. Antitumor efficacy studies were performed in female nude mice bearing MDA-MB-468 tumors, in which nanoparticles were administered intravenously twice a week for the duration of three weeks. Biodistribution of drug-containing nanoparticles in extracted tumors were examined, as well as reverse phase protein array (RPPA) analysis to gain insights into site-specific synergy. Results: Nanoparticles were spherical, with an average diameter of 9 nm. Both rapamycin and paclitaxel loaded favorably, allowing for customization of different ratios within nanoparticles. Combination indices demonstrated that a 3:1 ratio of RAP:PTX had the most synergy in MDA-MB-468 breast cancer cells in vitro, a synergy found to be preserved in vivo. Significant tumor regression (> 1.5 fold reduction from initial tumor volume) was observed in vivo upon administration of 3:1 RAP:PTX (15:5 mg/kg) nanoparticles. The precise ratio of rapamycin and paclitaxel (3:1) was found maintained in tumors 24 h after administration, an effect not achievable with free drug formulations. RPPA analysis demonstrated effective blocking of mTOR and Akt 24 h after administration of nanoparticles, key events in drug synergy. Discussion: Site-specific delivery of synergistic agents in precisely-controlled drug ratios, possible through their incorporation into nanoparticles, was shown to be highly efficacious against breast tumors. Findings demonstrate the ability to deliver specific drug ratios to tumors, potentially precluding the need to administer maximal doses of both agents in order to achieve synergy, lessening patient side-effects. This study demonstrates the potential for prediction of in vivo therapeutic outcomes from in vitro synergistic findings. Nanoparticle delivery of drugs may also yield enhanced understanding of mechanisms of synergy between molecular-targeted drugs and traditional chemotherapeutics in vivo, resulting in novel and more efficacious treatment regimens. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-03.

405TiP - A Phase II Study of Albumin-Bound Paclitaxel Combined with Epirubicin and Cyclophosphamide as Neo-Adjuvant Therapy in Breast Cancer Women

ABSTRACT Purpose To observe whether the efficacy and safety of Albumin-Bound Paclitaxel has advantage over Cremophor-Formulated Paclitaxel as neo-adjuvant therapy in breast cancer Patients and methods Twenty-six breast cancer patients were enrolled into Albumin-Bound Paclitaxel group. Albumin-Bound Paclitaxel 260mg/m2, Epirubicin 60 mg/m2, Cyclophosphamide 500mg/m2 was administrated as neo-adjuvant therapy. Twenty-six patients who were treated with Cremophor-Formulated Paclitaxel 175mg/m2, Epirubicin 60 mg/m2, Cyclophosphamide 500mg/m2 as the control group. The objective observation include the pathologic complete response rates, clinical response rates, safety and toxicity. The PI3K, pAKT, mTOR, BAD protein were examined before and after chemotherapy in two groups. Results 34.6% patients (9/26) in Albumin-Bound Paclitaxel group achieved a clinical complete response, this rate was 11.5% higher than control group which rate was 23.1%(6/26). 15.4% patients (4/26) in Albumin-Bound Paclitaxel group achieved a pathologic complete response, this rate was 11.6% higher than control group which rate was 3.8%(1/26). None patients (0/26) in Albumin-Bound Paclitaxel group occurs neutropenia but 15.4% patients (4/26) in control group occurs neutropenia during chemotherapy. All patients have normal left ventricular ejection fraction range (LVEF > 50%) before and after therapy. The positive rate of PI3K, mTOR, pAKT expression decreased 45.6%,42.5%,41.1% respectively in Albumin-Bound Paclitaxel group after chemotherapy and the control group decreased 13.2%,14.1%,10.1%. The positive rate of BAD expression increased 21.6% in Albumin-Bound Paclitaxel group after chemotherapy and the control group only increased 6.2%. Conclusion Albumin-Bound Paclitaxel has advantage in clinical response rate over Cremophor-Formulated Paclitaxel in breast cancer. Albumin-Bound Paclitaxel has no more additional adverse events than Cremophor-Formulated Paclitaxel. Albumin-Bound Paclitaxel has lower incidence of neutropenia than Cremophor-Formulated Paclitaxel. Albumin-Bound Paclitaxel decrease the expression of PI3K, pAKT, mTOR protein and increase the expression of BAD protein more significantly than Cremophor-Formulated Paclitaxel after chemotherapy Disclosure All authors have declared no conflicts of interest.

Abstract 3357: Targeting cancer stem cells by an anti-DLL4 antibody inhibits epithelial-to-mesenchymal transition, delays tumor recurrence and overcomes drug resistance in breast and pancreatic cancer

Abstract Accumulating evidence suggests that tumor growth, recurrence and metastasis are driven by a subset of highly tumorigenic cells referred to as cancer stem cells (CSCs) or tumor initiating cells. Several investigators have demonstrated that CSCs are relatively resistant to chemotherapy and that tumor recurrence and the development of drug resistance after chemotherapy are mediated by residual cancer stem cells. Furthermore, it has been demonstrated that the process of epithelial-to-mesenchymal transition (EMT) contributes to drug resistance and results in cells with CSC-like characteristics. We previously demonstrated that targeting Notch signaling pathway by a novel anti-DLL4 antibody inhibited tumor growth and decreased cancer stem cell frequency. Using patient-derived breast cancer and pancreatic cancer xenograft tumors we found that residual tumors after conventional chemotherapy (paclitaxel for breast cancer and gemcitabine for pancreatic cancer) were enriched with cancer stem cells. Gene expression analysis of cell populations enriched for CSCs revealed that several mesenchymal-associated genes were up-regulated, whereas epithelial gene was down-regulated. As shown by in vivo limiting dilution analyses, treatment with anti-DLL4 antibody decreased cancer stem cell frequency. Anti-DLL4 reversed chemotherapy-induced mesenchymal gene expression and delayed tumor recurrence. The residual tumor cells treated with conventional therapy eventually developed acquired drug resistance following repeated treatment. Compared to the parental xenograft, the resistant tumors had increased cancer stem cell frequency and tumorigenic ability. Treatment with anti-DLL4 antibody inhibited growth of these resistant tumors and re-sensitized them to the chemotherapeutic agent. This effect was in part due to the ability of the antibody to decrease cancer stem cell frequency in resistant tumors. The combination of antibody and chemotherapeutic agent further decreased cancer stem cell frequency compared to the parental tumor. Gene expression analysis demonstrated that resistant tumors had increased expression of many genes associated with EMT, multidrug resistance, DNA repair, and the Notch and Wnt pathways. Importantly, treatment with anti-DLL4 suppressed many gene expression changes in chemo-resistant tumors and resulted in a gene expression profile more like the original (drug sensitive) tumor. Immunohistochemical analysis indicated that anti-DLL4 mediated anti-tumor effect was associated with inhibition of EMT and induction of differentiation markers. Our findings provide a rationale to target cancer stem cells through interference with Notch pathway as a therapeutic approach in patients who are refractory to chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3357. doi:1538-7445.AM2012-3357

Microtubule‐binding protein CLIP‐170 is a mediator of paclitaxel sensitivity

CLIP-170 is a microtubule-binding protein and participates in diverse microtubule-associated cellular activities by regulating microtubule dynamics. Here we provide evidence that CLIP-170 is a mediator of the sensitivity of the anti-microtubule drug paclitaxel in breast cancer. In vitro cell proliferation assays reveal that CLIP-170 expression in breast cancer cell lines correlates with their sensitivity to paclitaxel. In addition, CLIP-170 expression in clinical samples of breast cancer correlates with the pathological response of tumours to paclitaxel-containing chemotherapy. Mitotic index and caspase-3 activity analyses reveal that CLIP-170 increases the abilities of paclitaxel to block cell cycle progression at mitosis and to induce apoptosis in breast cancer cells. By microtubule sedimentation assay and binding affinity analysis, we further find that CLIP-170 promotes paclitaxel binding to microtubules. In vitro tubulin polymerization assay shows that CLIP-170 enhances the activity of paclitaxel to promote microtubule assembly. These results demonstrate that CLIP-170 mediates paclitaxel sensitivity in breast cancer via a microtubule-dependent mechanism.

Regression of a meningioma during paclitaxel and bevacizumab therapy for breast cancer

Meningiomas often are not amenable to total resection, and subtotally resected meningiomas often recur. Recurrent meningiomas present a clinical dilemma, particularly when surgical and radiotherapy options are exhausted, as there are no chemotherapeutic options that demonstrate consistent, significant efficacy. We report a patient with the regression of a recurrent World Health Organization grade I meningioma during combination chemotherapy with bevacizumab and paclitaxel for breast cancer. This chemotherapy regimen has never been explored for recurrent meningiomas. While further data are necessary, we suggest that combination chemotherapy with bevacizumab and paclitaxel may be an option for treatment of recurrent meningiomas when no further surgical or radiotherapy options exist.

P1-13-04: Phase II Study of Topical Imiquimod and Abraxane for Treatment of Breast Cancer Cutaneous Metastases.

Abstract Background: Breast cancer (BC) cutaneous lesions can present as local chest wall recurrence or isolated sites of metastatic disease. Current treatments with full thickness chest wall resection, radiation therapy and chemotherapy are not curative; and have significant morbidity and poor overall response rates. Combining local immunomodulation and systemic chemotherapy may be more effective in treating cutaneous disease. Topical imiquimod (IMQ), a TLR-7 agonist, has shown clinical activity against cutaneous metastasis. Pre-clinical studies have shown IMQ to stimulate Th1 cytokine secretion and up-regulate immune co-stimulatory molecules at the tumor site; resulting in augmented tumor specific T cell immunity and tumor growth inhibition. Use of paclitaxel in BC, has demonstrated immunostimulatory effects of increased serum IFN-γ and enhanced NK/LAK cell activity. Abraxane (albumin-bound paclitaxel) may be used in conjunction with IMQ as steroid pre-treatment is not required. We hypothesize the immune effects of Abraxane may synergize and augment the IMQ anti-tumor effects, resulting in greater clinical response. A phase II single-arm study of chemoimmunotherapy with topical IMQ and Abraxane was initiated to determine its safety and therapeutic efficacy; and examine its effect on augmenting endogenous tumor specific immunity and inducing tumor molecular alterations associated with inhibition of tumor growth and/or common pathways of BC immune escape. Materials and Methods: Up to 15 BC patients with cutaneous lesions no longer amenable to standard therapy are enrolled and receive 3 treatment cycles. A treatment cycle consist of topical 5% IMQ to target lesions 4 days/week (wk.) and Abraxane 100 mg/m2 on Days 1, 8, 15 every 28 Days. Toxicity is evaluated per CTCAE v3.0 on Days 1, 8, 15 of each cycle and wks. 13, 16, 20, 24. Target lesion antitumor activity is assessed per modified WHO criteria (Complete response (CR); Partial response (PR); Stable disease (SD); Progressive disease (PD)) at baseline, wks. 4, 8, 12, 16, 20, 24. 2-mm target lesion skin biopsies are obtained pre-and post-treatment for histologic analysis and RT-PCR analysis of a 7 IFN-related gene signature associated with tumor inhibition. Immunity to HER2, IGFBP-2, TOPO-IIα, p53 and serum TGF-β levels are evaluated at baseline and wks. 12, 24 with IFN-γ ELISPOT and ELISA, respectively. Results: 10 patients have been enrolled. Median (range) values include: age, 54 years (48-92), time from metastatic diagnosis, 134 months (58-728), prior chemotherapy regimens, 5 (2-10). 5/10 patients had received prior local therapy, e.g., radiation. 5/10, 4/10, and 2/10 patients had triple negative, HER2+ and ER+/PR+ tumors, respectively. In 5 patients completing 3 treatment cycles, overall response rate (ORR) = 100% (3 CR, 2 PR). In the 5 patients who completed 1–2 treatment cycles, ORR = 80% (2 PR, 2 SD, 1 PD). Treatment related toxicity is primarily grade I/II neutropenia, anemia; grade I skin toxicity. Immunologic analyses are ongoing and will be presented with completed clinical data on all patients. Conclusions: Chemoimmunotherapy with topical IMQ and Abraxane is well-tolerated and shows excellent clinical efficacy in treating metastatic cutaneous lesions in heavily pretreated BC patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-13-04.

Abstract P4-08-06: Companion Diagnostic Test for Expression of β-III tubulin (ß3T) in Breast Cancer (BC) Using an Immunohistochemical (IHC) Assay in Studies of Ixabepilone (Ixa)

Abstract Background: Preclinical and retrospective clinical data suggest that ≥3T is a biomarker for poor prognosis and resistance to paclitaxel in breast cancer (BC). Preclinical studies have demonstrated that Ixa has activity in ≥3T over-expressing xenograft models resistant to taxanes and Ixa has shown clinical activity in BC patients with high ≥3T mRNA levels [Horak et al., J Clin Oncol 27:15s, 2009 (suppl; abstr 3587]. Dako and BMS have developed an IHC assay for detection of ≥3T to explore the potential of ≥3T as a biomarker to select BC patients that may benefit from Ixa. A neoadjuvant BC study comparing ixa to weekly paclitaxel employs this assay to assess ≥3T expression levels in patients’ tumor samples. Here we describe the application and initial validation testing of the assay in BC tumor samples. Methods: An IHC assay employing a murine monoclonal anti-≥3T antibody (TUJ1) was developed to assess ≥3T expression in formalin-fixed paraffin-embedded tissue specimens. Banked specimens for initial testing included 233 cases of invasive BC. Results: Patterns of ≥3T staining were typically homogeneous cytoplasmic staining with normal tissue elements such as endothelial cells and nerve cells serving as internal positive controls. Staining intensity was scored as 0, 1+, 2+ or 3+ with 0 being no visible staining and 2+ being equal to that of the internal positive control. Tumors with ≥50% cells staining ≥2+ were defined as positive for ≥3T based on previous published studies. Of the 233 breast tumors, 81 (35%) were positive and 152 (65%) were negative for ≥3T expression. HER2 status was defined for 128 of these specimens, with 37 of them (29%) found to be HER2 positive. Comparison of the samples with defined HER2 status demonstrated similar patterns of ≥3T expression, with 15 (40.5%) of the HER2+ samples being positive for ≥3T and 31 (34.1%) of HER2- specimens expressing elevated levels of ≥3T. In pathologist training exercises the inter-reader and intra-reader agreement of ≥3T scoring exceeded 85%. Antigen stability has been confirmed and other assay validation efforts are ongoing. Conclusions: These findings are consistent with previously reported data on ≥3T expression in breast cancer, and support the use of this ≥3T IHC assay in studies with ixabepilone. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-06.

Prospective randomized phase II study determines the clinical usefulness of genetic biomarkers for sensitivity to primary chemotherapy with paclitaxel in breast cancer

In patients with breast cancer, taxane as well as anthracycline play central roles in systemic chemotherapy. By evaluating the pathological response, we can gauge sensitivity to primary chemotherapy. However, biomarkers that would predict a response to taxane have not yet been established. We conducted a prospective randomized trial to evaluate whether selecting patients using sensitivity testing based on the gene expression of the tumor might enhance the probability of the pathological response. Five genes were identified as biomarkers derived from a microarray of DNA gene profiles from microdisected breast tumors. In the experimental arm (B1), 12 cycles of weekly paclitaxel, 80 mg/m(2) , were preoperatively given when the sensitivity test was positive and therefore judged to be sensitive to paclitaxel. When the test was negative, meaning insensitive to paclitaxel, four cycles of FEC100 were given (arm B2). In the control arm (A), paclitaxel was administered weekly without the use of the sensitivity test. A total of 92 patients were enrolled and 86 patients were analyzed. The pathological response rate (pRR) of each arm was 36.4% in B1 (expected sensitive to paclitaxel), 21.1% in A (control) and 12.5% in B2, respectively. Weekly paclitaxel-treated patients selected by the sensitivity test did not enhance the pRR. The study failed to validate sensitivity testing using five gene expressions for primary chemotherapy with paclitaxel in patients with breast cancer. However, this study suggests that a randomized phase II study is a robust tool for obtaining a rapid conclusion on the usefulness of biomarkers and could be the foundation for further large clinical trials.

The effects of chemotherapeutic drugs on VEGF secretion of solid tumors.

e13123 Background: The effects of chemotherapeutic drugs on angiogenesis are not well known. Recently, treatment with the lower doses of chemotherapeutic drugs, called metronomic chemotherapy, has been widely used in daily practice. One of the main objectives for this strategy is the antiangiogenic effects of the drugs at the lower doses. In this study we aimed to investigate the effects of chemotherapeutic drugs on serum VEGF levels of advanced tumors. We also tested the effects of different doses of widely used chemotherapeutic drugs on VEGF secretion of tumor cell lines. Methods: Thirty chemonaive patients with advanced colon cancer, breast cancer, or gastric cancer were included in the study. The treatment regimens were single-agent docetaxel or paclitaxel for breast cancer, docetaxel cisplatinum and 5-florouracil combination for gastric cancer, and either FOLFIRI or FOLFOX-4 regimens for colon cancer patients. Serum VEGF levels, taken just before the first and third cycles of chemotherapy, were assayed by sandwich ELISA. A human colon cancer cell line (HT-29), a breast cancer cell line (MCF-7), and a primary gastric cancer cell line established in our lab were used for in vitro tests. IC50 values were estimated by using MTT assay of varying doses of doxorubicine, 5-florouracil, irinotecan, oxaliplatin, docetaxel, and paclitaxel. The different doses of the drugs were tested for the efficacy on VEGF secretion of tumor cell lines. Results: A positive correlation between the tumor response rate and significant decrease in serum VEGF levels was found. The mean VEGF levels following 72 hours of incubation were 800 pg/mg protein for HT-29 cell line, 48 pg for MCF-7, and 240 pg for primary gastric cancer cell line. The test drugs yielded significantly more decrease in VEGF levels at the doses of -2log of IC50 in MCF-7 and primary gastric cancer cell lines than the dose of -1log and above. However, the same doses did not cause any significant change in VEGF secretion of HT-29 colon cancer cell line. Conclusions: The results of the current study suggest that the antiangiogenic effects of conventional chemotherapeutic drugs might have a role in the efficacy of the treatment. The effective metronomic doses of the chemotherapeutic drugs could be different in various tumors. No significant financial relationships to disclose.

Molecular Pharmacology and Antitumor Activity of PHT-427, a Novel Akt/Phosphatidylinositide-Dependent Protein Kinase 1 Pleckstrin Homology Domain Inhibitor

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity.

Increased Nuclear Localization of Transcription Factor Y-Box Binding Protein 1 Accompanied by Up-Regulation of P-Glycoprotein in Breast Cancer Pretreated with Paclitaxel.

Abstract Purpose: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein (P-gp) encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was performed to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer.Experimental Design: We evaluated the expression and localization of YB-1 and P-gp in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by EMSA. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay.Results: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-gp during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-gp. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-gp (P=0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (p< 0.001).Conclusions: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1141.

Abstract B139: Molecular pharmacology and antitumor activity of PHT-427 a novel AKT/PDPK1 pleckstrin homology domain inhibitor

Abstract The pleckstrin homology (PH) domain is a highly conserved three dimensional superfold found in many high valued pharmaceutical target proteins. While for the majority of PH domain proteins PtdIns binding is weak and non-specific, a subclass of approximately 40 PH domain containing proteins show high affinity for phosphatidylinositol-3-phosphates (PtdIns-3-P) causing their translocation to the plasma membrane and/or activation. Akt and PDKP1 are two such PH domain containing serine threonine kinase members of the PtdIns-3-kinase signaling pathway that plays a critical role in activating survival and anti-apoptotic signaling in cancer cells. This pathway is constitutively activated through mutation, overexpression and loss of the tumor suppressor PTEN in many different cancer types. There is emerging evidence that Akt and PDKP1 play complimentary yet independent roles in signaling by the pathway. Attempts to develop ATP catalytic site inhibitors of Akt and PDKP1 have generally resulted in toxic agents because of inhibition of off-target kinase family members. Through a process of reiterative molecular docking and structure refinement using a proprietary computational platform, and measurement of binding affinities by surface plasmon resonance (SPR) spectroscopy, we have developed inhibitors of these proteins that bind to the PH domain. We report here the development of PHT-427 a compound that binds to the PH domain of AKT and PDKP1. To develop PHT-427 an initial pharmacophore that bound to the PH domain PtdIns binding pocket was first identified. During the docking/modeling we identified a channel whithin the PH domains of both AKT and PDPK1 that could accommodate the binding of an alkyl chain. A series of analogs with C-4 to C-16 alkyl chain length were therefore synthesized and PHT-427 (C-12 chain) was found to bind with the highest affinity to the PH domains of both PDPK1 and AKT. PHT-427 gives a transient inhibition of AKT signaling in cells and tumor xenografts and a longer lasting inhibition of PDPK1 signaling. This inhibition correlated with decreased activation of downstream signaling targets for both proteins. PHT-427 given ias an oral formulation on a twice daily schedule inhibited the growth rate of human tumor xenografts in immunodeficient mice with up to an 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Tumors with a K-Ras mutation were less sensitive to PHT-427 as it has been seen for other inhibitors of the PtdIns-3-K signaling pathway. Combination antitumor studies showed that PHT-427 has greater than additive activity with paclitaxel in breast cancer and with erlotinib in non small cell lung cancer. There was minimal toxicity of PH-427 at doses that gave antitumor activity. PHT-427 given twice daily for 5 days caused no weight loss or change in blood chemistry. In summary we have identified a novel PH domain binding inhibitor of PDPK1/AKT signaling that has minimal toxicity and antitumor activity against human tumor xenografts.. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B139.

Delayed Seizure Associated with Paclitaxel‐Cremophor EL in a Patient with Early‐Stage Breast Cancer

Paclitaxel, a microtubule stabilizer, is an effective agent for treating cancer of the breast, ovary, head and neck, and lung. Because paclitaxel is insoluble in water, it is formulated with the micelle-forming Cremophor EL. Neurologic toxicity is well described with both the drug and this carrier, with most toxicities manifesting as peripheral neuropathy, motor neuropathy, autonomic neuropathy, and myopathy. Toxic effects on the central nervous system, such as seizures or encephalopathy, have been rarely reported; however, the seizures reported were closely related to the time of infusion. We describe a 41-year-old woman with no history of seizures who was treated with paclitaxel for breast cancer. Four days after the drug was infused, she developed a generalized tonic-clonic seizure that could not be attributed to other causes. The patient was treated with phenytoin and was able to complete her adjuvant chemotherapy with nab-paclitaxel without further events. Her condition was neurologically stable without phenytoin for the next 6 months. Use of the Naranjo adverse drug reaction probability scale indicated a possible association (score of 3) between the delayed seizure and paclitaxel or its solvent, Cremophor EL. Clinicians should be aware of the potential for seizure activity in patients who receive paclitaxel formulated with Cremophor EL.

Tau expression as a predictive marker in ovarian cancer patients (pts) treated with platinum/paclitaxel chemotherapy

5529 Background: Ovarian cancer is the most lethal gynecologic cancer and its standard treatment consists of cytoreductive surgery followed by paclitaxel/platinum-based chemotherapy. Additional prognostic biomarkers may allow for more tailored chemotherapy and an improved outcome. Recently, microtubule associated protein tau was identified as a marker of response to paclitaxel in breast cancer. Its predictive value in ovarian cancer has not been established. We evaluated the relevance of tau expression in ovarian cancer. Methods: Tissue specimens were obtained from 74 ovarian cancer patients (stage I-IV) who underwent cytoreductive surgery followed by standard paclitaxel/platinum (cisplatin or carboplatin) chemotherapy. Tau expression was determined by immunohistochemistry using semiquantitative DAKO test. Statistical analysis included long rank test and Mann Whitney test. Results: Median age of pts was 54 years (range 31–73). 25.7% and 74.3% of the pts were classified as tau-negative and tau-positive, respectively. In the univariate analysis 2-year progression free survival in tau-negative and tau-positive groups was 75.0% and 30.6%, respectively (p = 0.0153). This relationship was confirmed in the multivariate analysis including age, disease stage, histology type, and grade (HR 2.68; 95%CI [1.13–6.37]; p = 0.0255). Among the 46 pts with measurable disease, overall response rate in tau-negative and tau-positive groups was 90.9 and 54.5%, respectively (p = 0.0299, Z = 2.17). Conclusions: Tau protein expression seems to be a strong predictor of clinical outcome in ovarian cancer pts applied paclitaxel/platinum-based chemotherapy. No significant financial relationships to disclose.

Parkin regulates paclitaxel sensitivity in breast cancer via a microtubule‐dependent mechanism

Parkin is an E3 ubiquitin ligase encoded by the Parkin gene (also called PARK2, located at 6q25.2-q27) and is involved in the pathogenesis of Parkinson's disease and the development of cancer. Recently, Parkin has been demonstrated to interact with the microtubule cytoskeleton. However, the biological implication of the Parkin-microtubule axis has been poorly explored. In this study, we report for the first time that Parkin modulates sensitivity of the chemotherapeutic agent paclitaxel in breast cancer, via a microtubule-dependent mechanism. Our data reveal that Parkin binds to the outer surface of microtubules and increases paclitaxel-microtubule interaction, resulting in enhanced paclitaxel-induced microtubule assembly and stabilization. Our data further show that Parkin promotes the activity of paclitaxel to trigger multinucleation and apoptosis, rendering breast cancer cells more sensitive to this drug. Moreover, Parkin expression correlates with the pathological response of tumours to preoperative paclitaxel-containing chemotherapy. In addition, expression of Parkin also correlates with the sensitivity of paclitaxel in primary cultures of breast cancer cells. Our results identify Parkin as a novel mediator of paclitaxel sensitivity in breast cancer. In addition, our study suggests that patients harbouring tumours with high Parkin level would be more likely to benefit from paclitaxel-containing regimens.

Prolonged Dose-Dense Epirubicin and Cyclophosphamide Followed by Paclitaxel in Breast Cancer Is Feasible

Purpose We conducted a pilot study of dose-dense epirubicin/cyclophosphamide (EC) × 6 → paclitaxel (P) × 6 with pegfilgrastim. A previous dose-dense trial of FEC (5-fluorouracil [5-FU]/EC) × 6 with filgrastim → by weekly paclitaxel alternating with docetaxel × 18 was not feasible because of pneumonitis (with dose-dense FEC) and pericardial/pleural effusion (taxane phase). Dose-dense EC (without the 5-FU) is not associated with pneumonitis, and dose-dense paclitaxel (alone) is feasible. Primary objective was feasibility. Patients and Methods Patients with resectable breast cancer were enrolled, regardless of surgery status, tumor size, or nodal status. Treatment regimen consisted of every-2-week EC (100/600 mg/m 2) × 6 → by 2-weekly P (175 mg/m 2) × 6 with pegfilgrastim 6 mg on day 2. Results Between November 2004 and May 2005, 38 patients were enrolled. The median age was 47 years (range, 30-72 years); 33 of 38 (87%) were treated in the adjuvant setting and 27 of 33 (81%) had involved nodes (range, 1-46); 5 of 38 (13%) were treated pre-operatively; 33 of 38 (87%) completed all chemotherapy as planned; the remaining patients (13%) had treatment modifications for toxicity. Febrile neutropenia occurred in 6 of 38 patients (16 %) and only during EC. There were 12 hospitalizations in 9 of 38 patients (24%) enrolled. Conclusion Dose-dense every-2-week EC × 6 → P × 6 with pegfilgrastim is feasible based on our prospective definition.