Increased Paclitaxel Sensitivity Research Articles

SYK Blockade Enhances Paclitaxel Response in Ovarian Cancer

Abstract Inactivation of SYK synergistically increases paclitaxel sensitivity in ovarian cancer cells.

MicroRNA-101 inhibits cell progression and increases paclitaxel sensitivity by suppressing MCL-1 expression in human triple-negative breast cancer.

Triple-negative breast cancer is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of both miR-101 and MCL-1 in the sensitivity of human triple-negative breast cancer (TNBC) to paclitaxel. We found that the expression of miR-101 was strongly decreased in triple-negative breast cancer tissues and cell lines. The expression of miR-101 was not associated with clinical stage or lymph node infiltration in TNBC. Ectopic overexpression of miR-101 inhibit growth and induced apoptosis in vitro and suppressed tumorigenicity in vivo. MCL-1 was significantly overexpressed in most of the TNBC tissues and cell lines. Luciferase assay results confirmed MCL-1 as a direct target gene of miR-101. MiR-101 inhibited MCL-1 expression in TNBC cells and transplanted tumors. There was a negative correlation between the level of expression of miR-101 and MCL-1 in TNBC tissues. Suppression of MCL-1 enhanced the sensitivity of MDA-MB-435 cells to paclitaxel. Furthermore, miR-101 increased paclitaxel sensitivity by inhibiting MCL-1 expression. Our findings provide significant insight into the molecular mechanisms of TNBC carcinogenesis and may have clinical relevance for the development of novel, targeted therapies for TNBC.

Elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment.

Accumulating evidence has suggested that myristoylated alanine-rich C-kinase substrate (MARCKS) is critical for regulating multiple pathophysiological processes. However, the molecular mechanism underlying increased phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) and its functional consequence in neoplastic disease remain to be established. Herein, we investigated how phospho-MARCKS is regulated in breast carcinoma, and its role in the context of chemotherapy. In a screen of patients with breast tumors, we find that the abundance of phospho-MARCKS, not MARCKS protein per se, increased in breast cancers and positively correlated with tumor grade and metastatic status. Among chemotherapeutic agents, mitotic inhibitors, including paclitaxel, vincristine or eribulin, notably promoted phospho-MARCKS accumulation in multiple breast cancer cells. We further show that phospho-MARCKS acted upstream of Src activation upon paclitaxel exposure. Reduction of phospho-MARCKS by knockdown of MARCKS or pharmacological agents increased paclitaxel sensitivity. Particularly, a known phospho-MARCKS inhibitor, MANS peptide, was demonstrated to increase paclitaxel efficacy and attenuate angiogenesis/metastasis of xenografted breast cancer cells by decreasing abundance of phospho-MARCKS and messages of inflammatory mediators. Our data suggest that unresponsiveness of breast cancer to paclitaxel treatment is, at least in part, mediated by phospho-MARCKS and also provide an alternative therapeutic strategy against breast cancer by improving taxanes sensitivity.

Zerumbone inhibits growth of hormone refractory prostate cancer cells by inhibiting JAK2/STAT3 pathway and increases paclitaxel sensitivity.

Zerumbone, a phytochemical isolated from Zingiber zerumbet has been shown previously to exhibit antineoplastic activity. But, the effect of zerumbone in prostate cancer has not been evaluated. Prostate cancer is frequently associated with elevated levels of interleukin-6 (IL-6), which exerts its oncogenic effects through activation of Janus kinase 2 (JAK2) followed by activation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Here, we investigated whether the anticancer effects of zerumbone are mediated through inhibition of the JAK2/STAT3 signaling pathway and whether zerumbone can increase the paclitaxel (PTX) sensitivity of prostate cancer cells. Zerumbone exerted significant cytotoxicity of DU145 versus PC3 prostate cancer cells through cell cycle arrest at G0/G1 phase followed by apoptosis. Zerumbone selectively inhibited JAK2 in both DU145 and PC3 cells. However, the biological axis of IL-6/JAK2/STAT3 was inhibited only in DU145 cells as no STAT3 phosphorylation was detected in PC3 cells even after IL-6 stimulation. Other signaling pathways in DU145 cells remained unaffected. The expression of prostate cancer-associated genes, including cyclin D1, IL-6, COX2, and ETV1, was blocked. Zerumbone also synergistically increased the sensitivity to PTX. Further preclinical study might reveal the potential use of zerumbone as a chemotherapeutic agent for hormone refractory prostate cancer where IL-6/JAK2/STAT3 signaling is aberrantly active and may be combined with PTX.

HMSH2 expression is associated with paclitaxel resistance in ovarian carcinoma, and inhibition of hMSH2 expression invitro restores paclitaxel sensitivity.

The objective of the present study was to investigate the association between paclitaxel resistance, gene copy number, and gene expression in ovarian carcinoma, and to restore paclitaxel sensitivity in a paclitaxel-resistant ovarian carcinoma cell line by using hMSH2-targeting siRNA. Paclitaxel-resistant ovarian carcinoma cell lines OC3/TAX300 and OC3/TAX50 and their parental cell lines were analyzed by comparative genomic hybridization, and the expression levels of hMSH2 in ovarian carcinoma cell lines and tissues were determined. An siRNA targeted to hMSH2 mRNA was used to transfect a paclitaxel-resistant cell line. We assessed the morphological features, proliferation, and susceptibility to apoptosis of the transfected cells after paclitaxel treatment. Chromosome 2p21(gene locus of hMSH2) was amplified in OC3/TAX300 cells. hMSH2 was overexpressed in 93.9 and 47.6% of paclitaxel-treated and untreated ovarian carcinoma tissue samples(P=0.0001), respectively. hMSH2 was overexpressed in 93.3 and 54.2% of low-differentiated and moderate-to-highly differentiated ovarian carcinoma tissue samples(P=0.0008), respectively. hMSH2 expression was inhibited in the OC3/TAX300 cells transfected with hMSH2 siRNA. hMSH2 siRNA increased paclitaxel sensitivity, inhibited OC3/TAX300 cell proliferation(G2/M arrest), and increased susceptibility to apoptosis. hMSH2 expression was upregulated in ovarian carcinoma cell lines and tissues after paclitaxel treatment. hMSH2 overexpression is related to paclitaxel resistance and poor prognosis. Inhibition of hMSH2 expression invitro restores paclitaxel sensitivity in paclitaxel‑resistant ovarian carcinoma cell lines and indicates a new direction in adjuvant therapy for ovarian carcinoma.

Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.

The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

PD173074, a selective FGFR inhibitor, reverses MRP7 (ABCC10)-mediated MDR

Multidrug resistance protein 7 (MRP7, ABCC10) is a recently identified member of the ATP-binding cassette (ABC) transporter family, which adequately confers resistance to a diverse group of antineoplastic agents, including taxanes, vinca alkaloids and nucleoside analogs among others. Clinical studies indicate an increased MRP7 expression in non-small cell lung carcinomas (NSCLC) compared to a normal healthy lung tissue. Recent studies revealed increased paclitaxel sensitivity in the Mrp7−/− mouse model compared to their wild-type counterparts. This demonstrates that MRP7 is a key contributor in developing drug resistance. Recently our group reported that PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could significantly reverse P-glycoprotein-mediated MDR. However, whether PD173074 can interact with and inhibit other MRP members is unknown. In the present study, we investigated the ability of PD173074 to reverse MRP7-mediated MDR. We found that PD173074, at non-toxic concentration, could significantly increase the cellular sensitivity to MRP7 substrates. Mechanistic studies indicated that PD173074 (1μmol/L) significantly increased the intracellular accumulation and in-turn decreased the efflux of paclitaxel by inhibiting the transport activity without altering expression levels of the MRP7 protein, thereby representing a promising therapeutic agent in the clinical treatment of chemoresistant cancer patients.

Paclitaxel isomerisation in polymeric micelles based on hydrophobized hyaluronic acid

Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (Mw=15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelle's interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX.

Inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.

Paclitaxel (PAC) is the first-line chemotherapy drug for most breast cancer patients, but clinical studies showed that some breast cancer patients were insensitive to PAC, which led to chemotherapy failure. It was reported that Notch1 signaling participated in drug resistance of breast cancer. Here, we show whether Notch1 expression is related to PAC sensitivity of breast cancer. We employed Notch1 siRNA and Notch1 inhibitor, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT), to down regulate Notch1 expression in human breast cancer cells MDA-MB-231, and detected the inhibition effect by Western blotting and reverse trans cription-polymerase chain reaction, respectively. After 24 hours exposure to different concentration of PAC (0, 1, 5, 10, 15, 20, and 25 µg/ml), the viability of the control group and experimental group cells was tested by MTT. We also examined the expression of Notch1 in PAC sensitive and nonsensitive breast cancer patients, respectively by immunohistochemistry (IHC). The PAC sensitivity of breast cancer patients were identified by collagen gel droplet embedded culture-drug sensitivity test (CD-DST). Down regulation of Notch1 expression by Notch1siRNA interference or Notch1 inhibitor increased the PAC sensitivity in MDA-MB-231 cells (P < 0.05). Also, the expression of Notch1 in PAC sensitive patients was much lower than that of PAC non-sensitive patients (P < 0.01). Notch1 expression has an effect on PAC sensitivity in breast cancer patients, and the inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.

Human cancer cell line microRNAs associated with in vitro sensitivity to paclitaxel

Paclitaxel is a mainstay of treatment for many solid tumors, and frequently, clinical outcome is influenced by paclitaxel sensitivity. Despite this, our understanding of the molecular basis of paclitaxel response is incomplete. Recently, it has been shown that microRNAs (miRNAs) influence messenger RNA (mRNA) transcriptional control and can contribute to human carcinogenesis. In the present study, our objective was to identify miRNAs associated with cancer cell line response to paclitaxel and to evaluate these miRNAs as therapeutic targets to increase paclitaxel sensitivity. We measured the expression of 335 unique miRNAs in 40 human cancer cell lines selected from the NCI panel. We then integrated miRNA expression data with publicly available paclitaxel-sensitivity (GI50) data for each of the 40 cell lines to identify miRNAs associated with paclitaxel sensitivity. Ovarian cancer cell lines with differential miRNA expression and paclitaxel sensitivity were transiently transfected with miRNA precursors and inhibitors, and the effects on in vitro cell paclitaxel sensitivity were evaluated. Pearson’s correlation identified 2 miRNAs (miR-367 and miR-30a-5p) associated with the NCI40 cell line in vitro paclitaxel response (P<0.0003). Ovarian cancer cells were selected based on the association between paclitaxel sensitivity and miR-367/miR-30a-5p expression. Overexpression of miR-367 in the paclitaxel-sensitive cells [PA1; IC50, 1.69 nM, high miR-367 (2.997), low miR-30a-5p (−0.323)] further increased paclitaxel sensitivity, whereas miR-367 depletion decreased paclitaxel sensitivity. In contrast, overexpression and depletion of miR-30a-5p in the paclitaxel-resistant cells [OVCAR4; IC50, 17.8 nM, low miR-367 (−0.640), high miR-30a-5p (3.270)] decreased and increased paclitaxel sensitivity, respectively. We identified and successfully targeted miRNAs associated with human cancer cell line response to paclitaxel. Our strategy of integrating in vitro miRNA expression and drug sensitivity data may not only aid in the characterization of determinants of drug response but also in the identification of novel therapeutic targets to increase activity of existing therapeutics.

A CDK4/6 inhibitor enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring mutant KRAS as well as wild-type KRAS

The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15–30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma.

Abstract 5433: Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells.

Abstract Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of &amp;gt;300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P&amp;lt;0.05) of human ovarian cancer xenografts in two ovarian cancer models (Hey A8 and A2780); even greater growth inhibition was observed when PFKFB2 siRNA treatment was combined with paclitaxel. Cell growth inhibition in cell culture was linked to G1 arrest and to a modest increase in apoptosis. Induction of p21Cip1 and p27Kip1 protein by PFKFB2 knockdown contributed to the G1 arrest, since silencing p21Cip1 and p27Kip1 dramatically reduced PFKFB2 knockdown-induced G1 arrest. PFKFB2 knockdown also modulated the expression of CDK2 and the phosphorylation of p27Kip1. Induction of p53 protein contributed to both PFKFB2 knockdown-induced G1 arrest and to apoptosis, since silencing p53 markedly alleviated PFKFB2 knockdown-induced G1 arrest and apoptosis. PFKFB2 knockdown inhibited expression of the ABC transporter family members ABCC4 and ABCG2 at the level of transcription, possibly contributing to enhanced paclitaxel retention and increased cytotoxicity after PFKFB2 knockdown. Taken together, these results suggest that PFKFB2 protein may regulate cell growth and sensitivity to paclitaxel by multiple mechanisms, particularly in p53 wild type ovarian cancers. As methods to deliver siRNA evolve in the clinic, PFKFB2 may provide a novel target for enhancing sensitivity to paclitaxel-based chemotherapy in patients with low grade serous ovarian cancer where p53 function remains intact. Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in 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 5433. doi:10.1158/1538-7445.AM2013-5433

Promoter Methylation status of HIN-1 associated with outcomes of ovarian clear cell adenocarcinoma

BackgroundThis study is to analyze promoter methylation of various tumor suppressor genes in different types of ovarian carcinoma and to identify potential therapeutic targets of ovarian clear cell adenocarcinoma (OCCA).Materials and methodsThe promoter methylation statuses of 40 genes in primary ovarian carcinomas including 47 clear- and 63 non-clear-cell type tissues, 6 OCCA cell lines, 29 benign ovarian endometriotic cysts, and 31 normal controls were analyzed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The MS-MLPA results were correlated with clinicopathological features and outcomes of 47 OCCA patients. Functions of the target genes were further explored by Western Blot Analysis, apoptosis assay, and caspase-3/7 activity analysis.ResultsFrequencies of methylated RASSF1A, CDH13, CACNA1A, HIN-1, and sFRP5 genes in OCCA tissues were significantly higher than those in non-OCCA cancerous tissues and benign endometriotic cysts. The expected OS for patients with methylated promoters of HIN-1 was significantly worse than those for patients without methylated HIN-1 (30% vs. 62%, p = 0.002). The HIN-1 gene was over-expressed in ES2 cells, a significant reduction in cell growth and induction of apoptosis, and increasing paclitaxel sensitivity by reducing phosphorylation of Akt were observed.ConclusionsMethylation of HIN-1 promoter is a novel epigenetic biomarker associated with poor outcomes in OCCA patients. Ectopic expression of the HIN-1 gene increased paclitaxel sensitivity which is partly through Akt pathway.

β3 integrin modulates transforming growth factor beta induced (TGFBI) function and paclitaxel response in ovarian cancer cells

BackgroundThe extracellular matrix (ECM) has a key role in facilitating the progression of ovarian cancer and we have shown recently that the secreted ECM protein TGFBI modulates the response of ovarian cancer to paclitaxel-induced cell death.ResultsWe have determined TGFBI signaling from the extracellular environment is preferential for the cell surface αvß3 integrin heterodimer, in contrast to periostin, a TGFBI paralogue, which signals primarily via a ß1 integrin-mediated pathway. We demonstrate that suppression of ß1 integrin expression, in ß3 integrin-expressing ovarian cancer cells, increases adhesion to rTGFBI. In addition, Syndecan-1 and −4 expression is dispensable for adhesion to rTGFBI and loss of Syndecan-1 cooperates with the loss of ß1 integrin to further enhance adhesion to rTGFBI. The RGD motif present in the carboxy-terminus of TGFBI is necessary, but not sufficient, for SKOV3 cell adhesion and is dispensable for adhesion of ovarian cancer cells lacking ß3 integrin expression. In contrast to TGFBI, the carboxy-terminus of periostin, lacking a RGD motif, is unable to support adhesion of ovarian cancer cells. Suppression of ß3 integrin in SKOV3 cells increases resistance to paclitaxel-induced cell death while suppression of ß1 integrin has no effect. Furthermore, suppression of TGFBI expression stimulates a paclitaxel resistant phenotype while suppression of fibronectin expression, which primarily signals through a ß1 integrin-mediated pathway, increases paclitaxel sensitivity.ConclusionsTherefore, different ECM components use distinct signaling mechanisms in ovarian cancer cells and in particular, TGFBI preferentially interacts through a ß3 integrin receptor mediated mechanism to regulate the response of cells to paclitaxel-induced cell death.

Abstract 3057: CDK5 modulates the cell cycle, apoptosis and paclitaxel sensitivity in human ovarian cancer cells with wild-type p53 function

Abstract Cyclin-dependent kinase 5 (CDK5) is a ubiquitously expressed serine/threonine kinase that was initially identified in bovine brain extracts and plays an important role in normal neuron development and neurodegenerative diseases. This molecule has recently been shown to possess many cellular functions in non-neuronal tissues as well. Using a kinome siRNA screen, we identified CDK5 as a kinase that modulates paclitaxel sensitivity in human ovarian cancer cells. The aim of this report is to identify mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancer cells. Despite nucleotide sequence homology with human CDK1, a role of CDK5 in regulating the cell cycle remains unclear. Here we demonstrate dual roles of CDK5 in regulating the cell cycle and apoptosis. We have found that knockdown of CDK5 with multiple siRNAs markedly inhibits cell proliferation and increases paclitaxel sensitivity in several p53 wild type ovarian cancer cell lines using assays for cell viability and clonogenic growth. CDK5 knockdown with or without paclitaxel treatment induces G1 arrest of the cell cycle and apoptotic cell death linked to a significant induction of p53, p21Cip1 and p27Kip1 proteins. Induction of G1 arrest following CDK5 knockdown depends on induction of p21Cip1 and p27Kip1, since silencing p21Cip1 and/or p27Kip1 dramatically reduces CDK5 knockdown-induced G1 arrest. A CDK5 knockdown-induced increase of p53 expression contributes both to CDK5 knockdown-induced G1 arrest and to apoptosis, as silencing p53 markedly diminishes CDK5 knockdown-induced G1 arrest and apoptosis. As expected, a CDK5 knockdown-induced increase in p21Cip1 expression depends on p53-mediated transcriptional activation demonstrated by QRT-PCR and reporter luciferase assays. CDK5 knockdown activates caspase-3 and a pan-caspase inhibitor completely blocks CDK5 knockdown-induced apoptosis. Significant Inhibition of Bcl-2 expression is found in CDK5 knockdown-induced apoptosis and additional inhibition of Bcl-2 expression is found with combined CDK5 and paclitaxel treatment. Taken together, these data suggest that CDK5 can modulate paclitaxel sensitivity through regulating both the cell cycle and apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells with wild-type p53 function, providing a novel approach for enhancing the efficacies of paclitaxel-based regimens for low grade ovarian cancer in the clinic. 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 3057. doi:1538-7445.AM2012-3057

Deciphering the role of the ERCC2 gene polymorphism on anticancer drug sensitivity

ERCC2 [Xeroderma pigmentosum (XP) group D] belongs to the nucleotide excision repair pathway. It is also part of the TFIIH transcription complex and is required for the association of the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex with TFIIH. Using the NCI-60 panel of human tumor cell lines, we had shown that the ERCC2 gene variant Gln(751) was significantly associated to increased taxanes sensitivity and decreased ERCC2 gene expression. Since TFIIH is involved in both DNA repair and cell cycle progression, we hypothesized that quantitative or qualitative ERCC2 alterations might cause CAK liberation, allowing its activation of the G(2)/M transition. Enhancing mitosis entry would lead to hypersensitivity to spindle poisons, explaining the effect of ERCC2 polymorphisms on taxane sensitivity. Starting from ERCC2-deficient XP6BE, we generated several isogenic clones differing only by the Lys751Gln variation. Wild-type and variant ERCC2-expressing clones recovered ultraviolet radiation and cisplatin resistance but presented similar sensitivity to paclitaxel, demonstrating that the amino acid change was not involved in paclitaxel differential sensitivity in the NCI-60 panel. Using small interfering RNA approach, we knocked down ERCC2 expression and observed a block in the G(2)/M phase, with a consistent increase in paclitaxel sensitivity and no change in cisplatin sensitivity. We observed in addition an increase in CDK1 activity, as evaluated by histone H1 phosphorylation. We evaluated messenger RNA (mRNA) half-life in the isogenic lines and observed a more rapid degradation in cells bearing the variant construct. We concluded that the increased paclitaxel sensitivity of ERCC2 variant cell lines is a consequence of lower gene expression, likely due to decreased stability of the variant ERCC2 mRNA.

Identification of Markers of Taxane Sensitivity Using Proteomic and Genomic Analyses of Breast Tumors from Patients Receiving Neoadjuvant Paclitaxel and Radiation

To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies. Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR). Proteomic and validation immunohistochemical analyses revealed that alpha-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery. We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane-based therapy.

NAC-1, a potential stem cell pluripotency factor, contributes to paclitaxel resistance in ovarian cancer through inactivating Gadd45 pathway

Nucleus accumbens-1 (Nac1 or NAC-1) belongs to the BTB/POZ transcription factor family and is a novel protein that potentially participates in self-renewal and pluripotency in embryonic stem cells. In human cancer, NAC-1 is upregulated in several types of neoplasms, but particularly in recurrent chemoresistant ovarian carcinomas, suggesting a biological role for NAC-1 in the development of drug resistance in ovarian cancer. We have assessed this possibility and demonstrated a correlation between NAC-1 expression and ex vivo paclitaxel resistance in ovarian serous carcinoma tissues and cell lines. We found that expression of Gadd45gamma-interacting protein 1 (Gadd45gip1), a downstream target negatively regulated by NAC-1, was reduced in paclitaxel-resistant cells. Ectopic expression of NAC-1 or knockdown of Gadd45gip1 conferred paclitaxel resistance, while NAC-1 knockdown or ectopic expression of Gadd45gip1 increased paclitaxel sensitivity. Furthermore, silencing NAC-1 expression or disrupting NAC-1 homodimerization by a dominant negative NAC-1 protein that contained only the BTB/POZ domain induced expression of Gadd45gamma which interacted with Gadd45gip1. Reducing Gadd45gamma expression by shRNAs partially enhanced paclitaxel resistance. Thus, this study provides new evidence that NAC-1 upregulation and homodimerization contribute to tumor recurrence by equipping ovarian cancer cells with the paclitaxel-resistant phenotype through negative regulation of the Gadd45 pathway.

Estrogen Receptor Expression and Sensitivity to Paclitaxel in Breast Cancer

A retrospective analysis of CALGB trial 9344 suggested paclitaxel administration following cyclophosphamide and doxorubicin adjuvant chemotherapy is most beneficial for patients with ER? negative (ER?-) breast cancer. Since the cytotoxic effects of paclitaxel are cell cycle dependent, we postulated that the relationship between ER? and the effectiveness of adjuvant paclitaxel reflects the observation that ER? positive (ER?+) breast cancers proliferate more slowly than ER?- breast cancers. Three in vitro models (MCF-7, T47D and ZR-75) were examined to compare growth rates and paclitaxel-induced apoptosis in ER?+ and ER?- clones of the same, originally ER?+ cell line. For the T47D and ZR-75 cell lines, loss of ER? was associated with a decrease in doubling time and an increase in paclitaxel sensitivity. However, when cell culture conditions were altered to achieve equivalent cell proliferation rates, no difference in paclitaxel sensitivity was observed. Similarly, an ER?- clone of MCF-7 cells that did not exhibit an enhanced growth rate compared to its ER?+ counterpart also did not show increased paclitaxel sensitivity. The combined apoptotic effects of tamoxifen and paclitaxel on MCF-7 cells were not synergistic or even clearly additive. In these in vitro models, the effectiveness of paclitaxel correlated more closely with growth rate than ER? expression. These data suggest that measurements of tumor proliferation may provide more accurate predictive markers for the benefits of adjuvant paclitaxel than ER? analysis.