Paclitaxel-induced Cytotoxicity Research Articles

Tibetan tea extract exerts anti-tumor effects and potentiate paclitaxel-induced cytotoxicity and apoptosis in human hepatocellular carcinoma cells HepG2

Tibetan Tea (TT), traditionally esteemed by indigenous communities on the Qinghai-Tibet Plateau for over thirteen centuries, is a post-fermented tea recognized for its wide spectrum of biological functions. These include antioxidant capabilities, hypocholesterolemic effects, anti-hyperglycemic properties, and immune system enhancement. Nevertheless, additional medicinal potentials of TT, including its anticancer effects, remain to be unveiled in empirical studies. We aimed to evaluate the antiproliferative and chemotherapy-enhancing effects of TT extract on human hepatocellular carcinoma (HCC) cells, thereby initiating an inquiry into the potential molecular mechanisms involved. In this study, it was observed that TT extract suppressed the proliferation of HCC cells and triggered significant apoptosis. This apoptotic induction was facilitated through the activation of caspase-3, caspase-8, and caspase-9, along with decreased expression ratio of Bcl-2/Bax protein in HCC cells. Moreover, the combinational treatment of TT extract and Paclitaxel (Taxol) was found to enhance the anticancer effect. The expression level of P-glycoprotein (P-gp) encoded by the MDR1 (Multiple Drug Resistance) gene was remarkably decreased, revealing that TT extract might have the potential to be utilized as an adjuvant to promote HCC treatment. In conclusion, the Tibetan Tea extract exhibits cytotoxic, anticancer, and chemosensitization properties.

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

Forskolin affects proliferation, migration and Paclitaxel-mediated cytotoxicity in non-small-cell lung cancer cell lines via adenylyl cyclase/cAMP axis

Non-Small-Cell Lung Cancer (NSCLC) is considered one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Despite the undoubted therapeutic advances that have occurred in clinical practice over time, due to its high degree in both heterogeneity and resistance, NSCLC remains largely incurable. As a natural cAMP elevating agent, Forskolin has shown anti-cancer properties in different tumor types, thus supposing its possible usage in treating malignancies. In this study, we investigated the Forskolin outcome in H1299 and A549 NSCLC cell lines, either alone or in combination with Paclitaxel. We proved that Forskolin impairs cell growth and migration ability of these cells, concurrently. Albeit with a different extent between H1299 and A549, changes in cell-cycle progression and epithelial-mesenchymal markers were observed in response to Forskolin administration. Interestingly, comparable cell growth impairment was also obtained with the cAMP phosphodiesterase inhibitor IBMX, while the employment of adenylyl cyclase inhibitor SQ22536 counteracted, at least in part, the Forskolin-mediated anticancer effects. Besides as a single agent, we also demonstrated that Forskolin strongly enhances Paclitaxel-induced cytotoxicity, affecting cell death mainly via apoptosis induction. Notably, H89-mediated protein kinase A (PKA) inhibition further deteriorated the combination outcome. Altogether, our data designate Forskolin as a possible anticancer molecule in NSCLC, and recognize the adenylyl cyclase/cAMP axis as one of the pathways involved in. Although achieved at preclinical stage, our findings encourage the design of future studies aimed at further exploring the Forskolin employment in NSCLC treatment.

Paclitaxel-Containing Extract Exerts Anti-Cancer Activity through Oral Administration in A549-Xenografted BALB/C Nude Mice: Synergistic Effect between Paclitaxel and Flavonoids or Lignoids.

Taxus yunnanensis is a paclitaxel-containing herb with traditional usage in cancer treatment, and its extract possesses great oral bioavailability of paclitaxel. However, it is elusive whether paclitaxel-containing extract (HDS-1) can exert anti-tumor effect through oral administration and how other components contribute to its efficacy. Therefore, we investigate the oral-route anti-tumor effect of HDS-1 in A549-bearing mice. HDS-1-derived flavonoids (HDS-2) and lignoids (HDS-3) are hypothesized to contribute to HDS-1's efficacy, and their effects of enhancing enterocytic absorption and cytotoxicity of paclitaxel are validated in 2 permeability experiments and apoptosis-related assay, respectively. In vivo, A549 growth is significantly inhibited by 86.1 ± 12.94% (P < 0.01) at 600 mg/kg of HDS-1 and 65.7 ± 38.71% (P < 0.01) at 200 mg/kg. HDS-2 and HDS-3 significantly reduce the efflux ratio of paclitaxel to 2.33 and 3.70, respectively, in Caco-2 permeability experiment and reduce paclitaxel reflux in MDCK-MDR1 experiment. Furthermore, HDS-2 and HDS-3 potentiated paclitaxel-induced cytotoxicity by 19.1–22.45% (P < 0.05) and 10.52–18.03% (P < 0.05), respectively, inhibited the expression of cyclinB1, Bcl-2, and pMCL-1, and increased the percentage of necrosis cell in the condition of paclitaxel exposure. Conclusively, paclitaxel-containing extracts exert anti-cancer effects through oral administration, and flavonoid and lignoids contribute to its anti-cancer effect through simultaneously improving enterocytic absorption of paclitaxel and the cytotoxic effect of paclitaxel.

Morphine counteracts the effects of paclitaxel in triple-negative breast cancer cells.

Several studies have provided evidence that opioids may play a role in cancer recurrence and metastasis. Multiple research data indicate that morphine can act as a proliferative or suppressive agent on tumour cells depending on the applied concentration. Therefore, this study was aimed to investigate whether the presence of clinically relevant concentrations of morphine has any effect on the efficacy of paclitaxel, a widely used chemotherapeutic drug, on the viability and apoptosis of human triple-negative breast cancer cell line. MDA.MB.231 cells were treated with paclitaxel in the presence or absence of morphine and examined for cell proliferation by the MTT assay. In addition, the effect of morphine on paclitaxel-induced apoptosis was investigated by flow cytometric assay and by the ratio of Bax/Bcl-2 mRNA expression levels with quantitative real-time (qRT)-PCR. Morphine significantly increased the proliferation of breast cancer cells at low concentrations (0.1-2.5 μM) but higher concentrations showed cytotoxic effect. Pre-treatment with 0.1 or 1 μM of morphine decreased the paclitaxel-induced cytotoxicity, the proportion of apoptotic cell, and the ratio of Bax/Bcl-2 mRNA expressions. Our data suggest that morphine promotes breast cancer cell viability at clinically relevant plasma concentrations and reduces the apoptotic effect of paclitaxel. This interaction may be very important in clinical settings; however, more studies are needed to explore the plausible mechanisms of interaction and to correlate such findings through in vivo animal studies as well as clinically.

Exposure to low intensity ultrasound removes paclitaxel cytotoxicity in breast and ovarian cancer cells

BackgroundPaclitaxel (Taxol) is a microtubule-stabilizing drug used to treat several solid tumors, including ovarian, breast, non-small cell lung, and pancreatic cancers. The current treatment of ovarian cancer is chemotherapy using paclitaxel in combination with carboplatin as a frontline agent, and paclitaxel is also used in salvage treatment as a second line drug with a dose intensive regimen following recurrence. More recently, a dose dense approach for paclitaxel has been used to treat metastatic breast cancer with success.Paclitaxel binds to beta tubulin with high affinity and stabilizes microtubule bundles. As a consequence of targeting microtubules, paclitaxel kills cancer cells through inhibition of mitosis, causing mitotic catastrophes, and by additional, not yet well defined non-mitotic mechanism(s).ResultsIn exploring methods to modulate activity of paclitaxel in causing cancer cell death, we unexpectedly found that a brief exposure of paclitaxel-treated cells in culture to low intensity ultrasound waves prevented the paclitaxel-induced cytotoxicity and death of the cancer cells. The treatment with ultrasound shock waves was found to transiently disrupt the microtubule cytoskeleton and to eliminate paclitaxel-induced rigid microtubule bundles. When cellular microtubules were labelled with a fluorescent paclitaxel analog, exposure to ultrasound waves led to the disassembly of the labeled microtubules and localization of the signals to perinuclear compartments, which were determined to be lysosomes.ConclusionsWe suggest that ultrasound disrupts the paclitaxel-induced rigid microtubule cytoskeleton, generating paclitaxel bound fragments that undergo degradation. A new microtubule network forms from tubulins that are not bound by paclitaxel. Hence, ultrasound shock waves are able to abolish paclitaxel impact on microtubules.Thus, our results demonstrate that a brief exposure to low intensity ultrasound can reduce and/or eliminate cytotoxicity associated with paclitaxel treatment of cancer cells in cultures.

N-acylethanolamine-hydrolysing acid amidase: A new potential target to treat paclitaxel-induced neuropathy.

Although paclitaxel is an effective chemotherapeutic agent used to treat multiple types of cancer (e.g. breast, ovarian, neck and lung), it also elicits paclitaxel-induced peripheral neuropathy (PIPN), which represents a major dose-limiting side effect of this drug. As the endogenously produced N-acylethanolamine, palmitoylethanolamide (PEA), reverses paclitaxel-induced mechanical hypersensitivity in mice, the main goals of this study were to examine if paclitaxel affects levels of endogenous PEA in the spinal cord of mice and whether exogenous administration of PEA provides protection from the occurrence of paclitaxel-induced mechanical hypersensitivity. We further examined whether inhibition of N-acylethanolamine-hydrolysing acid amidase (NAAA), a hydrolytic PEA enzyme, would offer protection in mouse model of PIPN. Paclitaxel reduced PEA levels in the spinal cord, suggesting that dysregulation of this lipid signalling system may contribute to PIPN. Consistent with this idea, repeated administration of PEA partially prevented the paclitaxel-induced mechanical hypersensitivity. We next evaluated whether the selective NAAA inhibitor, AM9053, would prevent paclitaxel-induced mechanical hypersensitivity in mice. Acute administration of AM9053 dose-dependently reversed mechanical hypersensitivity through a PPAR-α mechanism, whereas repeated administration of AM9053 fully prevented the development of PIPN, without any evidence of tolerance. Moreover, AM9053 produced a conditioned place preference in paclitaxel-treated mice, but not in control mice. This pattern of findings suggests a lack of intrinsic rewarding effects, but a reduction in the pain aversiveness induced by paclitaxel. Finally, AM9053 did not alter paclitaxel-induced cytotoxicity in lung tumour cells. Collectively, these studies suggest that NAAA represents a promising target to treat and prevent PIPN. The present study demonstrates that the chemotherapeutic paclitaxel alters PEA levels in the spinal cord, whereas repeated exogenous PEA administration moderately alleviates PIPN in mice. Additionally, targeting NAAA, PEA's hydrolysing enzyme with a selective compound AM9053 reverses and prevents the PIPN via the PPAR-α mechanism. Overall, the data suggest that selective NAAA inhibitors denote promising future therapeutics to mitigate and prevent PIPN.

Administration with hyperoside sensitizes breast cancer cells to paclitaxel by blocking the TLR4 signaling

Breast cancer is a malignancy and one of the most frequent causes of cancer death among women worldwide. Paclitaxel is a common chemotherapeutic drug and has recently been shown to facilitate tumor cell escape during cytotoxic chemotherapy by inducing inflammatory mediators and pro-survival protein expression. Hyperoside is a flavonoid glycoside compound and exerts anti-inflammation, and anti-tumor growth properties. However, its function in breast cancer chemosensitivity remains poorly elucidated. In this study, hyperoside exhibited little cytotoxicity to normal human breast mammary epithelial cell lines, and also protected against paclitaxel-induced cytotoxicity in MCF-10A. Importantly, treatment with hyperoside engendered not only inhibition of cell viability, but also potentiated cancer cell sensitivity to paclitaxel in TLR4-positive breast cancer MDA-MB-231 cells by suppressing cell viability, and increasing cell apoptosis and caspase-3 activity. Nevertheless, although hyperoside exposure restrained cell viability, its treatment presented little effects to paclitaxel sensitivity in TLR4-null HCC1806 cells. Intriguingly, paclitaxel stimulation activated the TLR4-NF-κB signaling, which was reversed after hyperoside administration. Concomitantly, hyperoside also attenuated paclitaxel-mediated anti-apoptotic Bcl-2 expression, but enhanced the effects of paclitaxel on pro-apoptotic Bax expression, and pro-inflammatory cytokine IL-6 and IL-6 levels in MDA-MB-231 cells. Importantly, restoring the TLR4 pathway overturned hyperoside-evoked chemosensitivity to paclitaxel in MDA-MB-231 cells. Thus, hyperoside may elevate breast cancer cell sensitivity to paclitaxel by blocking TLR4 activation-mediated pro-inflammatory and pro-survival approaches, thereby endorsing its usefulness as a promising therapeutic combination to overcome chemosensitivity in breast cancer.

The microtubule targeting agents eribulin and paclitaxel activate similar signaling pathways and induce cell death predominantly in a caspase-independent manner

ABSTRACT Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

Histone Deacetylase Inhibitor, Trichostatin A, Synergistically Enhances Paclitaxel-Induced Cytotoxicity in Urothelial Carcinoma Cells by Suppressing the ERK Pathway.

Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces, inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA’s cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.

Long Non-coding RNA CDKN2B Antisense RNA 1 Gene Contributes to Paclitaxel Resistance in Endometrial Carcinoma.

Endometrial cancer (EC) is the most common malignancy of the female reproductive tract. In this study, we clarified the clinical significance of CDKN2B antisense RNA 1 (CDKN2B-AS) gene, and its effects on paclitaxel sensitivity in EC. Firstly, CDKN2B-AS gene was highly expressed in EC tissues and cell lines. The high-expression of CDKN2B-AS gene was associated with high pathological grade and low paclitaxel sensitivity of EC tissues. Knockdown of CDKN2B-AS gene sensitized Ishikawa/PA and HEC1A/PA cells to paclitaxel, and promoted paclitaxel-induced cytotoxicity. Secondly, the low-expression of miR-125a-5p was closely associated with low paclitaxel sensitivity of EC cells, and up-regulation of miR-125a-5p could increase paclitaxel sensitivity of Ishikawa/PA and HEC1A/PA cells. MiR-125a-5p also mediated the suppressive effects of knockdown of CDKN2B-AS on paclitaxel resistance in EC cells. Thirdly, B-cell lymphoma-2 (Bcl2) and Multidrug Resistance-Associated Protein 4 (MRP4) genes were target genes of miR-125a-5p, which modulated paclitaxel resistance of Ishikawa/PA and HEC1A/PA cells through targeted silencing Bcl2 and MRP4. In conclusion, high-expression of CDKN2B-AS is associated with a poor response to paclitaxel of EC patients, and knockdown of CDKN2B-AS inhibits paclitaxel resistance through miR-125a-5p-Bcl2/MRP4 pathway in EC patients. Our findings help elucidate the molecular mechanisms of chemoresistance in EC patients.

Bag-1 silencing enhanced chemotherapeutic drug-induced apoptosis in MCF-7 breast cancer cells affecting PI3K/Akt/mTOR and MAPK signaling pathways.

The multifunctional anti-apoptotic Bag-1 protein has important roles in apoptosis, proteasome-mediated degradation, transcriptional regulation, and intracellular signaling. Bag-1 promotes cell survival and proliferation, and is overexpressed in breast cancer. Therefore, Bag-1-targeted therapy might be a promising strategy to treat breast cancer. However, the effects of Bag-1 silencing in combination with conventional chemotherapeutic drugs on cell viability and major signaling pathways have not yet been fully investigated in breast cancer cells. In this study, we investigated the cytotoxic effects of Bag-1 silencing, alone and in combination with cisplatin or paclitaxel treatment, in MCF-7 breast cancer cells. Bag-1 knockdown by shRNA or siRNA transfection sensitized MCF-7 cells to apoptosis induced by cisplatin or paclitaxel. Combination of Bag-1 silencing and drug treatment more potently downregulated the pro-survival PI3K/Akt/mTOR and p44/42 mitogen activated protein kinase (MAPK) pathways, and more potently upregulated the stress-activated p38 and SAPK/JNK MAPK pathways. Bag-1-silenced drug-treated cells had also highly reduced proliferative capacity, downregulated cyclin-cyclin dependent kinase complexes and upregulated tumor suppressors p21 and Rb. These results overall indicated that Bag-1 silencing enhanced cisplatin- or paclitaxel-induced cytotoxicity through multiple pathways. In conclusion, Bag-1 targeted therapy might enhance the therapeutic potential of conventional anti-cancer drugs in the treatment of breast cancer.

MiR-193b-3p possesses anti-tumor activity in ovarian carcinoma cells by targeting p21-activated kinase 3

miR-193b-3p was found to be downregulated and contributed to ovarian cancer (OC) progression. In the present study, we aimed to study the detailed role of miR-193b-3p in the development of OC and the underlying molecular mechanism. The results showed that miR-193b-3p was downregulated while PAK3 was upregulated in OC cells. Ectopic expression of miR-193b-3p and PAK3 knockdown repressed cell proliferation, promoted paclitaxel-induced cytotoxicity, and reinforced paclitaxel-mediated caspase-3 activity increase in OC cells. Notably, miR-193b-3p was identified to directly target PAK3 and suppressed PAK3 expression. Moreover, enforced expression of PAK3 partially overturned the effects of miR-193b-3p on OC cell proliferation and paclitaxel sensitivity. In conclusion, miR-193b-3p possessed anti-tumor activity in OC through inhibiting cell proliferation and enhancing paclitaxel sensitivity by targeting PAK3. Therefore, our study suggested that the miR-193b-3p/PAK3 axis might be a potential novel therapeutic target for OC.

Abstract 3856: The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells

Abstract The chemotherapeutic agent paclitaxel increases cancer cell microtubule stability, induces mitotic arrest and cancer cell death and improves survival of cancer patients. However, in ovarian and other cancers, it only elicits a response in less than half of patients who receive it. We have previously shown that enhancing microtubule stability in cells prior to paclitaxel treatment, further increases paclitaxel-induced microtubule stability and cancer cell death (Ahmed et al., Cancer Cell, 2007;12:514 and Ahmed et al., Cancer Research 2011;71:5806). We now report that the microtubule-associated protein CRMP2, and FER kinase are expressed in almost half of high-grade serous ovarian cancers and that inhibiting the phosphorylation of CRMP2 by FER is sufficient for enhancing paclitaxel-induced microtubule stability and cytotoxicity in multiple ovarian cancer cell lines. We are also able propose a detailed mechanism underlying these observations. A combination of biochemistry, super-resolution fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy and electron microscopy reveals that CRMP2 induces microtubule nucleation, elongation, bundling and stability. Moreover, FER phosphorylates CRMP2 at 6 tyrosine residues, two of which (Tyr479 and Tyr499) are critical for modulating CRMP2 function; this agrees with site-directed mutagenesis in cancer cells which points to Tyr479 as a critical site for association between CRMP2 and microtubules. A comparison of the crystal structures of wild-type CRMP2 and Tyr479Glu/Tyr499Glu phosphomimetic mutants indicate that phosphorylation of these sites induces significant conformational changes that prevent CRMP2 from forming tetramers that are known to be critical for its microtubule bundling activity. This then leads us to propose a molecular model of how CRMP2 associates with microtubules, explaining how targeting CRMP2 phosphorylation can be exploited for enhancing the therapeutic efficacy of paclitaxel. Citation Format: Yiyan Zheng, Ritika Sethi, Frank von Delft, Ahmed Ashour Ahmed. The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells. [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 3856.

Abstract 3100: Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity

Abstract Glucocorticoid receptor (GR) activity in triple-negative breast cancer (TNBC) is associated with cell survival and chemotherapy resistance. Targetable molecular drivers for TNBC have been difficult to identify and therefore standard treatment remains limited to chemotherapy. Recently, new-generation small molecule Hsp90 inhibitors (e.g. ganetespib and NVP-AUY922) have proven to be both potent inhibitors of Hsp90 client proteins and far less toxic than previous Hsp90 inhibitors. Moreover, favorable clinical responses have been observed in some TNBC patients enrolled in a phase II clinical trial of ganetespib monotherapy. A ganetespib and paclitaxel clinical trial is ongoing in breast cancer; however, it is not known which Hsp90 client proteins contribute to increased tumor chemotherapy sensitivity. Recent studies in our lab found that inhibition of glucocorticoid receptor (GR) activity plays an important role in Hsp90 inhibitor-mediated sensitization of TNBC cells to paclitaxel-induced cytotoxicity. However, we also observed that following significant shRNA-mediated GR depletion, ganetespib still provided some sensitization of TNBC cells to paclitaxel. This suggests that inactivation of additional anti-apoptotic Hsp90 client proteins play a role in mediating ganetespib effects. Proteins closely associated with GR function that are also key Hsp90 inhibitor client proteins include the androgen receptor (AR, implicated in TNBC proliferation and apoptosis) and JAK, a component of JAK/STAT signaling pathway implicated in TNBC tumorigenesis and chemoresistance. Here we hypothesize that AR and JAK/STAT signaling have overlapping but distinct roles in Hsp90-mediated chemotherapy sensitization of TNBCs. Western blot analysis of TNBC xenografts treated with ganetespib +/- paclitaxel showed depletion of AR as well as JAK1 and 2. Ganetespib treatment also led to decreased levels of TMPRSS2 gene expression, an AR target gene. Additionally Western blot analysis showed decreased levels of activated phosphorylated STAT3 transcriptional factor, a downstream mediator of JAK, following ganetespib treatment of TNBC cell lines. These results suggest the involvement of AR and JAK in mediating ganetespib mechanism of action. We are currently beginning to determine how AR and JAK/STAT signaling pathways may interact with GR signaling by establishing CRISPR/Cas9 knockouts of GR, AR, and/or JAK in TNBC cells treated with ganetespib +/- paclitaxel. Understanding the relative roles of Hsp90 client proteins in TNBC is predicted to lead to a more rational selection of patients with TNBCs likely to benefit from Hsp90-inhibitor treatment. Citation Format: Abena S. Agyeman, Wesley J. Jun, Suzanne D. Conzen. Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity. [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 3100.

Hsp90 Inhibition Results in Glucocorticoid Receptor Degradation in Association with Increased Sensitivity to Paclitaxel in Triple-Negative Breast Cancer.

Targetable molecular drivers for triple-negative breast cancer (TNBC) have been difficult to identify; therefore, standard treatment remains limited to conventional chemotherapy. Recently, new-generation small-molecule Hsp90 inhibitors (e.g., ganetespib and NVP-AUY922) have demonstrated improved safety and activity profiles over the first-generation ansamycin class. In breast cancer, clinical responses have been observed in a subset of TNBC patients following ganetespib monotherapy; however, the underlying biology of Hsp90 inhibitor treatment and tumor response is not well understood. Glucocorticoid receptor (GR) activity in TNBC is associated with chemotherapy resistance. Here, we find that treatment of TNBC cell lines with ganetespib resulted in GR degradation and decreased GR-mediated gene expression. Ganetespib-associated GR degradation also sensitized TNBC cells to paclitaxel-induced cell death both in vitro and in vivo. The beneficial effect of the Hsp90 inhibitor on paclitaxel-induced cytotoxicity was reduced when GR was depleted in TNBC cells but could be recovered with GR overexpression. These findings suggest that GR-regulated anti-apoptotic and pro-proliferative signaling networks in TNBC are disrupted by Hsp90 inhibitors, thereby sensitizing TNBC to paclitaxel-induced cell death. Thus, GR+ TNBC patients may be a subgroup of breast cancer patients who are most likely to benefit from adding an Hsp90 inhibitor to taxane therapy.

Pharmacoethnicity in Paclitaxel-Induced Sensory Peripheral Neuropathy.

Paclitaxel is used worldwide in the treatment of breast, lung, ovarian, and other cancers. Sensory peripheral neuropathy is an associated adverse effect that cannot be predicted, prevented, or mitigated. To better understand the contribution of germline genetic variation to paclitaxel-induced peripheral neuropathy, we undertook an integrative approach that combines genome-wide association study (GWAS) data generated from HapMap lymphoblastoid cell lines (LCL) and Asian patients. GWAS was performed with paclitaxel-induced cytotoxicity generated in 363 LCLs and with paclitaxel-induced neuropathy from 145 Asian patients. A gene-based approach was used to identify overlapping genes and compare with a European clinical cohort of paclitaxel-induced neuropathy. Neurons derived from human-induced pluripotent stem cells were used for functional validation of candidate genes. SNPs near AIPL1 were significantly associated with paclitaxel-induced cytotoxicity in Asian LCLs (P < 10(-6)). Decreased expression of AIPL1 resulted in decreased sensitivity of neurons to paclitaxel by inducing neurite morphologic changes as measured by increased relative total outgrowth, number of processes and mean process length. Using a gene-based analysis, there were 32 genes that overlapped between Asian LCL cytotoxicity and Asian patient neuropathy (P < 0.05), including BCR. Upon BCR knockdown, there was an increase in neuronal sensitivity to paclitaxel as measured by neurite morphologic characteristics. We identified genetic variants associated with Asian paclitaxel-induced cytotoxicity and functionally validated the AIPL1 and BCR in a neuronal cell model. Furthermore, the integrative pharmacogenomics approach of LCL/patient GWAS may help prioritize target genes associated with chemotherapeutic-induced peripheral neuropathy.

Inhibition of Spleen Tyrosine Kinase Potentiates Paclitaxel-Induced Cytotoxicity in Ovarian Cancer Cells by Stabilizing Microtubules

Resistance to chemotherapy represents a major obstacle for long-term remission, and effective strategies toovercome drug resistance would have significant clinical impact. We report that recurrent ovarian carcinomas after paclitaxel/carboplatin treatment have higher levels of spleen tyrosine kinase (SYK) and phospho-SYK. Invitro, paclitaxel-resistant cells expressed higher SYK, and the ratio of phospho-SYK/SYK positively associated with paclitaxel resistance in ovarian cancer cells. Inactivation of SYK by inhibitors or gene knockdown sensitized paclitaxel cytotoxicity invitro and invivo. Analysis of the phosphotyrosine proteome in paclitaxel-resistant tumor cells revealed that SYK phosphorylates tubulins and microtubule-associated proteins. Inhibition of SYK enhanced microtubule stability in paclitaxel-resistant tumor cells that were otherwise insensitive. Thus, targeting SYK pathway is a promising strategy to enhance paclitaxel response.

Silencing dishevelled-1 sensitizes paclitaxel-resistant human ovarian cancer cells via AKT/GSK-3β/β-catenin signalling.

Expression of dishevelled-1 (DVL1) has recently been linked to cancer progression, however, its role in resistance to cancer therapy is unclear. In this study, we aimed to explore the function of DVL1 in paclitaxel-resistant human ovarian cancer cells. The MTT assay was used to assess effects of DVL1 silencing on sensitivity of cells that were otherwise resistant to paclitaxel (Taxol). Western blotting and immunofluorescence staining were used to examine effects of DVL1 on AKT/GSK-3β/β-catenin signalling. Dishevelled-1 was found to be over-expressed in a paclitaxel-resistant cell line derived from human ovarian cancer cell line A2780 (A2780/Taxol line) as well as parental A2780 cells. Down-regulation of DVL1 (using the inhibitor 3289-8625 or siRNA (siDVL1) against DVL1) sensitized A2780/Taxol cells to paclitaxel. Over-expression of DVL1 in A2780 cells increased protein levels of P-gp, BCRP and Bcl-2, which are known targets of β-catenin. Silencing DVL1 in A2780/Taxol cells also reduced levels of these proteins, and led to accumulation of β-catenin. In addition, DVL1 aberrantly activated AKT/GSK-3β/β-catenin signalling. Inactivation of AKT signalling attenuated DVL1-mediated inhibition of GSK-3β and accumulation of β-catenin, in both A2780 and A2780/Taxol cells. Taken together, these results suggest that silencing DVL1 sensitized A2780/Taxol cells to paclitaxel, by down-regulating AKT/GSK-3β/β-catenin signalling, providing a novel strategy for chemosensitization of ovarian cancer to paclitaxel-induced cytotoxicity.