Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and β-Tubulin.

Photoaffinity labeling approaches have historically been used in pharmacology to identify molecular targets. This methodology has played a pivotal role in identifying drug-binding domains and searching for novel compounds that may interact at these domains. In this review we focus on studies of microtubule stabilizing agents of natural product origin, specifically taxol (paclitaxel).

Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence.

The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein.

Nanoparticle-Encapsulated Doxorubicin Demonstrates Superior Tumor Cell Kill in Triple Negative Breast Cancer Subtypes Intrinsically Resistant to Doxorubicin.

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemoreffactory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox.

Improved Dose-Response Relationship of (+)-Discodermolide-Taxol Hybrid Congeners.

(+)-Discodermolide is a microtubule-stabilizing agent with potential for the treatment of taxol-refractory malignancies. (+)-Discodermolide congeners containing the C-3'-phenyl side chain of taxol (paclitaxel) were synthesized based on computational docking models predicting this moiety would fill an aromatic pocket of β-tubulin insufficiently occupied by (+)-discodermolide, thereby conferring improved ligand-target interaction. It was recently demonstrated, however, that the C-3'-phenyl side chain occupied a different space, instead extending toward the M-loop of β-tubulin, where it induced a helical conformation, hypothesized to improve lateral contacts between adjacent microtubule protofilaments.

Expression of βV-Tubulin in Secretory Cells of the Fallopian Tube Epithelium Marks Cellular Atypia.

Objectives: Class V Beta tubulin isotype (βV-tubulin) was recently found to have tissue-specific expression patterns in epithelial tissues with secretory function and aberrant expression in tumors. The aims of this pilot study were (a) to examine expression of βV-tubulin in the fallopian tube epithelium (FTE) of patients who underwent salpingectomy, (b) to characterize FTE atypia in high-risk patients with BRCA mutations, and (c) to determine expression of βV-tubulin in serous ovarian neoplasms.

Methods: Immunohistochemistry, with a highly specific antibody developed in our laboratory against human βV-tubulin, was used to evaluate expression in paraffin-embedded sections of the fallopian tube (n = 82) and tumors (n = 13), from prospectively selected cases, categorized by reason for salpingectomy.

Therapeutic Advances and New Directions for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a molecularly diverse grouping with poor prognosis for which chemotherapy remains the foundation of treatment. The molecular heterogeneity of the disease rationalizes its diverse biological behavior and differential response to treatment. Estimates of up to 20% of patients diagnosed have germline mutations in DNA-damage repair-pathway genes, namely and and this can be used to select patients likely to respond to platinums and/or inhibitors of poly(ADP-ribose) polymerase (PARP).

A Novel Indication for Panobinostat as a Senolytic Drug in NSCLC and HNSCC.

Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death.

Structural Basis of Microtubule Stabilization by Discodermolide.

Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Using X-ray crystallography we elucidated the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32, in the taxane pocket of β-tubulin. The two compounds bind in a very similar hairpin conformation, as previously observed in solution.

Therapies for triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is a heterogeneous disease associated with a high risk of recurrence, and therapeutic options are currently limited to cytotoxic therapy. Germ-line mutations may occur in up to 20% of unselected patients with TNBC, which may serve as a biomarker identifying which patients may have tumors that are particularly sensitive to platinums and/or inhibitors of poly(ADP-ribose)polymerase. A substantial proportion of patients with TNBCs not associated with germ-line BRCA mutations may have tumors that are ‘BRCA-like’, rendering those individuals potential candidates for similar strategies.

N-cadherin/FGFR promotes metastasis through epithelial-to-mesenchymal transition and stem/progenitor cell-like properties.

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis.

Exploiting MEK inhibitor-mediated activation of ERα for therapeutic intervention in ER-positive ovarian carcinoma.

While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes.

High expression of class III β-tubulin predicts good response to neoadjuvant taxane and doxorubicin/cyclophosphamide-based chemotherapy in estrogen receptor-negative breast cancer.

Background: Expression of class ΙΙΙ β-tubulin (βΙΙΙ-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether βΙΙΙ-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy.

Characterization of a human βV-tubulin antibody and expression of this isotype in normal and malignant human tissue.

There are seven distinct β-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. βV-tubulin, like βIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function.

Insights into 4E-BP1 and p53 mediated regulation of accelerated cell senescence.

Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence.

Resistance to discodermolide, a microtubule-stabilizing agent and senescence inducer, is 4E-BP1-dependent.

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence.

Antitumor activity of vinflunine: effector pathways and potential for synergies.

Vinflunine is an innovative microtubule inhibitor of the vinca alkaloid class with distinct tubulin-binding properties. Preclinical evaluation of this novel microtubule inhibitor has shown superior antitumor activity against a broad spectrum of tumor types in vitro and in vivo, in comparison with other vinca alkaloids. The antitumor effect of vinflunine is largely attributable to its modulation of microtubule dynamics, and is mediated by its ability to induce apoptosis in target cells.

The effect of ketoconazole on the pharmacokinetics and pharmacodynamics of ixabepilone: a first in class epothilone B analogue in late-phase clinical development.

Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4.

Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone.

Targeting protein translation in human non small cell lung cancer via combined MEK and mammalian target of rapamycin suppression.

Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death.

Peripheral blood mononuclear and tumor cell pharmacodynamics of the novel epothilone B analogue, ixabepilone.

Background: We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken.

Patients And Methods: Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.

Potentiation of taxol efficacy and by discodermolide in ovarian carcinoma xenograft-bearing mice.

Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma.

Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation.

Enhancement of the therapeutic efficacy of taxol by the mitogen-activated protein kinase kinase inhibitor CI-1040 in nude mice bearing human heterotransplants.

Taxol may contribute to intrinsic chemoresistance by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cytoprotective pathway in human cancer cell lines and tumors. We have previously shown additivity between Taxol and the MEK inhibitor, U0126 in human cancer cell lines. Here, the combination of Taxol with an orally bioavailable MEK inhibitor, CI-1040, was evaluated in human lung tumors heterotransplanted into nude mice.

Phase I clinical and pharmacokinetic study of BMS-247550, a novel derivative of epothilone B, in solid tumors.

Purpose: The purpose of this study was to determine the maximum tolerated dose, toxicity, and pharmacokinetics of BMS-247550 administered as a 1-h i.v. infusion every 3 weeks.

Mechanisms of Taxol resistance related to microtubules.

Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients.

Validation of the pharmacodynamics of BMS-247550, an analogue of epothilone B, during a phase I clinical study.

The primary aims of this study were to evaluate the timecourse and dose response of microtubule bundle formationin peripheral blood mononuclear cells (PBMCs) and to correlate these data with BMS-247550 pharmacokinetics. The data presented here were obtained from 17 patients enrolled in a Phase I trial who received five dose levels of BMS-247550 (7.4-59.