Chemoresistance Factor Research Articles

Combination treatment with fulvestrant and various cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5‐fluorouracil) has a synergistic effect in estrogen receptor‐positive breast cancer

Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to chemotherapy and a limited survival benefit. We have previously shown a combination effect of taxanes and fulvestrant and suggested that this treatment may be useful for ER-positive breast cancer. In this study, we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents. In vitro, the effects of combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) and three hormone drugs (fulvestrant, tamoxifen, and 4-hydroxytamoxifen) were examined in ER-positive breast cancer cell lines using CalcuSyn software. Changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1, and microtubule-associated protein tau were also examined after exposure of the cells to hormone drugs. In vivo, tumor sizes in mice were evaluated after treatment with docetaxel or doxorubicin alone, fulvestrant alone, and combinations of these agents. Combination treatment with fulvestrant and all five chemotherapeutic agents in vitro showed synergistic effects. In contrast, tamoxifen showed an antagonistic effect with all the chemotherapeutic agents. 4-Hydroxytamoxifen showed an antagonistic effect with doxorubicin and 5-fluorouracil, but a synergistic effect with taxanes and vinorelbine. Regarding chemoresistant factors, Bcl2 and microtubule-associated protein tau were downregulated by fulvestrant. In vivo, a combination of fulvestrant and docetaxel had a synergistic effect on tumor growth, but fulvestrant and doxorubicin did not show this effect. In conclusion, fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents, and especially with docetaxel, in vitro and in vivo.

Biologic anticancer activity of designed ankyrin repeat proteins (darpins) targeted against dnmt1 and conjugated to docetaxel leads to eradication of chemoresistant mutated squamous cell epsophageal cancer stem cells

IntroductionCurrent anticancer therapies succeed at eradicating bulky disease but miss a tumour reservoir consisting of stem/progenitor cells (SCs) that lead to disease recurrence and metastasis. Tumour stem cells are characterised...

Abstract P4-01-01: Combination Treatment with Fulvestrant and Various Cytotoxic Agents Has a Synergistic Effect in ER-Positive Breast Cancer Cell Lines In Vitro and In Vivo.

Abstract Background: Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers since they can be treated with anti-estrogen therapies. However, ER-positive breast cancers have low sensitivity to chemotherapy and the survival benefitof chemotherapy is limited. Microtubule associated protein-tau (MAPT) plays an important role in taxane sensitivity. In a previous study we showed that ER signaling influenced MAPT expression and that fulvestrant, an estrogen receptor antagonist, downregulated MAPT and reversed resistance to taxane in MAPT-and ER-positive breast cancer cells (SABCS 2009, Abstract 2118). In clinical treatment for breast cancer, the advantages and disadvantages of combination treatment with endocrine therapy and chemotherapy have long been discussed. Several studies have shown that endocrine therapy has an antagonistic effect on chemotherapy, which suggests that concurrent use of a hormone drug and a chemotherapeutic agent should be avoided clinically. However, most of these studies used tamoxifen (TAM) and anthracycline regimens. The effects of combination treatment using new hormone therapies, such as aromatase inhibitors or fulvestrant, and chemotherapeutic agents have not been examined thoroughly. In this study we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents in vitro and in vivo. Methods: In vitro, the effects of different combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-FU) and three hormone drugs (TAM, 4-hydroxytamoxifen (4OH-TAM), and fulvestrant) were examined in ER-positive breast cancer cell lines (MCF-7, ZR75-1) using CalcuSyn software. The cells were also exposed to hormone drugs and changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1 (MRP1), and MAPT were examined. In vivo, xenografts were created using MCF-7 cells transplanted into BALB/c nu/nu mice. Tumor sizes were evaluated after the mice were treated with tamoxifen alone, fulvestrant alone, docetaxel alone, and combinations of these drugs. Results: Combination treatment with fulvestrant and all chemotherapeutic agents in vitro showed a synergistic effect regardless of the fulvestrant dose. In contrast, TAM showed an antagonistic effect with all the chemotherapeutic agents, with the strongest antagonism occurring at a low dose of TAM. 4OH-TAM showed an antagonistic effect with doxorubicin and 5-FU, and a synergistic effect with taxanes and vinorelbine. In the analysis of chemoresistant factors, Bcl2 and MAPT were downregulated by fulvestrant, but upregulated by TAM and 4OH-TAM. In vivo, the combination of fulvestrant and docetaxel showed the strongest inhibitory effect on tumor growth. Conclusion: TAM and 4OH-TAM had different effects in combination with chemotherapeutic agents. Fulvestrant downregulated ER and chemoresistant factors such as Bcl2 and MAPT, which are regulated via ER. Fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents in vitro and in vivo. These results suggest that combination therapy with fulvestrant and chemotherapeutic agents may be effective for ER-positive breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-01.

Bcl-2 and β1-integrin predict survival in a tissue microarray of small cell lung cancer

Introduction:Survival in small cell lung cancer (SCLC) is limited by the development of chemoresistance. Factors associated with chemoresistance in vitro have been difficult to validate in vivo. Both Bcl-2 and β1-integrin have been identified as in vitro chemoresistance factors in SCLC but their importance in patients remains uncertain. Tissue microarrays (TMAs) are useful to validate biomarkers but no large TMA exists for SCLC. We designed an SCLC TMA to study potential biomarkers of prognosis and then used it to clarify the role of both Bcl-2 and β1-integrin in SCLC.Methods:A TMA was constructed consisting of 184 cases of SCLC and stained for expression of Bcl-2 and β1-integrin. The slides were scored and the role of the proteins in survival was determined using Cox regression analysis. A meta-analysis of the role of Bcl-2 expression in SCLC prognosis was performed based on published results.Results:Both proteins were expressed at high levels in the SCLC cases. For Bcl-2 (n=140), the hazard ratio for death if the staining was weak in intensity was 0.55 (0.33–0.94, P=0.03) and for β1-integrin (n=151) was 0.60 (0.39–0.92, P=0.02). The meta-analysis showed an overall hazard ratio for low expression of Bcl-2 of 0.91(0.74–1.09).Conclusions:Both Bcl-2 and β1-integrin are independent prognostic factors in SCLC in this cohort although further validation is required to confirm their importance. A TMA of SCLC cases is feasible but challenging and an important tool for biomarker validation.

Elevated XIAP expression alone does not confer chemoresistance

Background:In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated.Methods:To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression.Results:Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits.Conclusion:Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.

Abstract 645: Class V β-tubulin expression as biomarker in solid tumors

Abstract Microtubules are a component essential of the cytoskeleton and mediate a complex network of functions, including the assembly of mitotic spindle but also the intracellular trafficking of signaling proteins. In order to achieve their functions, composition of microtubules is tightly regulated, particularly at the (+) end, exposing the β-tubulin subunit. At least seven independent genes encode for β-tubulin, whose expression is diverse in several tissues. Functionally β-tubulin isotypes can be divided in constitutive isotypes and regulatory isotypes. Regulatory isotypes include class III β-tubulin (TUBB3), whose role as factor of resistance to chemotherapy has been demonstrated in a large number of solid tumors. Some structural motifs of TUBB3 are shared with class V β-tubulin (TUBB6), but since no commercial antibodies are available for this isotype its role as an additional factor of chemoresistance is not known. In this work we have developed a polyclonal anti-TUBB6 antibody. Since the highest degree of dishomology between the β-tubulin isotypes is concentrated at the C-terminus, all the tails of the β-tubulin isotypes were cloned in E. coli in a carrier vector encoding MBP (maltose-binding protein). Recombinant proteins were expressed in E. coli and then purified through columns filled with Amylose resin column, able to entrap MBP. The negative control was represented by MBP alone. The developed anti-TUBB6 antibody was validated since it demonstrated specific reactivity only against TUBB6. Therefore, the profile of expression of TUBB6 was assessed in a panel of normal and cancer tissues. In normal tissues, immunostaining of TUBB6 was confined to given cellular elements and in a focal pattern such as in ciliary cells of bronchus, salpinx, thereby suggesting that class V is focally associated to the cilium complex. In contrast to the focal expression noticed in normal tissues, in cancer specimens TUBB6 exhibited a diffuse staining pattern, with the highest expression detectable in colon and prostate carcinoma. In order to have some insights on the role of TUBB6 as a biomarker, we studied TUBB6 expression in a cohort of 56 ovarian cancer patients with a median follow up of 54 months. There was no statistically significant difference in terms of Time to Progression (TTP) in cases with high versus low TUBB6 expression (p value= 0.4). On the other hand, cases with high TUBB6 expression showed a worse Overall Survival (OS) with respect to cases with low TUBB6 expression (median OS= 50 months versus median OS= 96 months, respectively, p value=0.047). In summarizing these results, patients expressing high levels of TUBB6 are featured by biological aggressiveness and poor response in terms of OS to standard chemotherapy. These results suggest that TUBB6 could be useful as a biomarker for ovarian cancer patients and possibly other tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 645.

Role of STAT3 decoy oligodeoxynucleotides on cell invasion and chemosensitivity in human epithelial ovarian cancer cells

Recent studies have reported that STAT3 activation is associated with poor prognosis in human epithelial ovarian cancer. STAT3 has been proposed to play an important role in ovarian cancer metastasis and chemoresistance. This mechanism, however, is still not thoroughly understood. In this study, to investigate the role of STAT3 on ovarian cancer cells, we used decoy oligodeoxynucleotide (ODN) technology to regulate STAT3 in SKOV3 and OVCAR3 cells in vitro. Cell invasive power and chemo-sensitivity were assessed in the cells transfected with STAT3 decoy ODN and control ODN. Western blot analysis was used to examine the expression of EMMPRIN, P-gp, and Akt. Results showed that STAT3 decoy ODN inhibited cancer cell invasive power and enhanced sensitivity to paclitaxel for SKOV3 and OVCAR3 cells. The mechanism involved the inhibition of EMMPRIN, P-gp, and pAkt by STAT3 decoy ODN. These three proteins were probably the target proteins of STAT3. These findings suggest that STAT3 is a key factor for ovarian cancer metastasis and chemoresistance. STAT3 decoy ODN may prove to be a beneficial therapeutic agent, especially for invasive or chemoresistant ovarian cancer.

Abstract A73: The Bcl-2/BclXL inhibitor, ABT263, enhances paclitaxel-induced apoptosis in NSCLC models

Abstract The anti-apoptotic protein, BclXL, is thought to be an important chemo-resistance factor (Amundsen et al, 2000), and the combination of a BclXL inhibitor and paclitaxel exhibits increased NSCLC cancer cell killing in culture and in xenograft models (Shoemaker et al, 2006). ABT263 is a small molecule inhibitor of Bcl-2, BclXL and Bcl-w, that is currently in clinical trials (Tse et al, 2008). We set out to address the following questions 1) is ABT-263 broadly synergistic with paclitaxel in NSCLC cells, 2) what is the mechanism of the synergistic interaction and 3) can we identify biomarkers to guide the selection of patients that are likely to respond to the combination of ABT263 and taxane based chemotherapy regimens? We screened a panel of 26 NSCLC cell lines for synergy with ABT263. All 26 cell lines exhibited a synergistic response to the combination, as evaluated by Bliss additivity, and 20 cell lines had Bliss sum values in excess of 100, considered to be a robust synergy response in this assay. In order to address the mechanism of synergy, we performed time-lapse microscopy on GFP-H2b expressing A549 cells. We find that the addition of ABT263 alters the timing and fate of cells that enter mitosis in the presence of paclitaxel. Paclitaxel alone caused prolonged mitotic arrest usually followed by mitotic slippage. ABT263 as a single agent had little effect on mitosis, but caused cells to die rapidly from a mitotic arrest state when added to paclitaxel. This resulted in an increase in the rate and percentage of cell death compared to either single agent. To identify biomarkers that might influence synergy between ABT263 and paclitaxel, we identified genes whose mRNA levels had significant correlations with the Bliss score. Together these data suggest that the combination of ABT263 and paclitaxel has the potential to have improved efficacy in cancers that are refractory to paclitaxel. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A73.

Inactivation of O 6-methylguanine-DNA methyltransferase in soft tissue sarcomas: association with K-ras mutations

The DNA-repair protein O(6)-methylguanine-DNA methyltransferase removes alkyl adducts from the O(6)-position of guanine. The adducts can mispair with T during DNA replication, resulting in a G-to-A mutation. Epigenetic inactivation of O(6)-methylguanine-DNA methyltransferase has been found in human neoplasia and is considered one of the implicated factors in chemoresistance. Sixty-two patients with soft tissue sarcomas were analyzed with regard to the status of O(6)-methylguanine-DNA methyltransferase protein expression status using immunohistochemistry and promoter hypermethylation of the MGMT gene using methylation-specific PCR. G-to-A transitions in codons 12 and 13 of the K-ras oncogene were investigated using PCR and direct automated sequencing analysis. A loss of O(6)-methylguanine-DNA methyltransferase expression was noted in 20 (32.3%) cases of 62 total soft tissue sarcomas. The MGMT promoter hypermethylation rate was 33.9% (21/62 cases). Of the 54 sarcomas evaluated, K-ras mutations were found in only 2 (3.7%) cases. Loss of O(6)-methylguanine-DNA methyltransferase expression and MGMT promoter hypermethylation showed a significant association with high American Joint Committee on Cancer stage, high French Federation of Cancer Centers grade, and aggressive behavior. On multivariate analysis, these were not an independently significant prognostic factors. However, when the group receiving chemotherapy was analyzed (n = 27), loss of O(6)-methylguanine-DNA methyltransferase expression was correlated with worse survival on multivariate analysis (P = .024). MGMT promoter hypermethylation status had a strong correlation with loss of O(6)-methylguanine-DNA methyltransferase expression (P = .000). Our results suggest that MGMT promoter hypermethylation and loss of O(6)-methylguanine-DNA methyltransferase expression tend to be associated with poor prognosis and that the loss of O(6)-methylguanine-DNA methyltransferase protein expression frequently occurs via MGMT promoter hypermethylation. However, MGMT promoter hypermethylation was not significantly associated with point mutations of K-ras at codons 12 and 13 in sarcomas.

Pharmacogenomics Approach Reveals MRP1 (ABCC1)-Mediated Resistance to Geldanamycins

Geldanamycin and its analogues belong to a new class of anticancer agents that inhibit the molecular chaperone heat shock protein 90. We hypothesized that membrane transporters expressed on tumor cells may contribute at least in part to cellular sensitivity to these agents. The purpose of this study is to identify novel transporters as determinant for sensitivity and resistance to geldanamycins. To facilitate a systematic study of chemosensitivity across multiple geldanamycin analogues, we correlated mRNA expression profiles of majority of transporters with anticancer drug activities in 60 human tumor cell lines (NCI-60). We subsequently validated the gene-drug correlations using cytotoxicity and transport assays. The GA analogues displayed negative correlations with mRNA expression levels of the multidrug resistance protein 1 (MRP1, ABCC1). Suppressing MRP1 efflux using the inhibitor MK-571 and small interfering RNA in cell lines with intrinsic and acquired MRP1 overexpression (A549 and HL-60/ADR) and in cell lines stably transduced with MRP1 (MCF7/MRP1) increased intracellular drug accumulation and increased tumor cell sensitivity to geldanamycin analogues. These results suggest that elevated expression of MRP1, like the alternative efflux transporter MDR1 (ABCB1, P-glycoprotein), can significantly influence tumor cell sensitivity to geldanamycins as a potential chemoresistance factor.

Gene Expression Profile Analysis According to Recurrent Gene Copy Number Abnormalities Defines a Diffuse Large B-Cell Lymphoma Subgroup Characterized by 9p21 Locus Deletion, Ribosome Machinery Deregulation and Poor Prognosis. A GELA Study

Genomic gains and losses play a crucial role in the development and progression of DLBCL. Some gains or losses are associated with particular morphologic or clinical manifestations and correlate with the “germinal center B-cell like” (GCB)/non-GCB phenotype, as defined by gene expression profiles (GEP). We previously developed a reliable and routinely single applicable PCR assay, which provided information regarding gain/loss of relevant genes, and prognosis in DLBCL, termed QMPSF (Multiplex PCR of Short Fluorescent Fragments). Here, we combined GEP and QMPSF approaches to delineate molecular pathways related to recurrent gene copy number abnormalities (GCNA) and assess their prognosis value in patients treated by R-CHOP. For this purpose a series of 69 newly diagnosed DLBCL, included in the 98–5 GELA trial with available tumor DNA was studied (median age = 69 years [59–79], IPI2–3: 64%; 4–5: 36%, 40 treated by R-CHOP and 29 by CHOP). A single QMPSF assay, validated by CGH array, to detect GCNA of 8 relevant genes including SIM1 (6q16), MYC (8q24), CDNK2A (9p21), RB1 (13q14), REL (2p13), BCL2 (18q21), TP53 (17p13), and CDKN1B (12p13) was performed. In addition a dedicated QMPSF assay that provides a “bar code” of the 9p21 locus containing CDKN2A (p16INK4a and p14ARF) and CDKN2B (p15INK4b) was designed. To delineate specific gene expression profile according to recurrent GCNA a subset of 52 patients were studied by both GEP (Affymetrix U133A) and QMPSF technologies. Gains of MYC, BCL2, and REL were observed in 13, 28 and 20 % respectively. DNA copy losses of TP53, CDNK2A, RB1 and SIM1 were observed in 9, 40, 6 and 17 % of cases respectively. Using supervised analysis, we delineated specific GEP according to the most frequent GCNA detected by QMPSF. Interestingly, a signature related to 9p21 locus (CDKN2A/CDKN2B) deletion was associated with an overexpression of several ribosome machinery coding genes and the involvement of distinct antiapoptotic molecular mechanisms. Subsequent genomic analysis with the dedicated assay indicated that in most of cases deletions were homozygous and abolished simultaneously p14arf and p16INK4a expression. With a median follow-up of 81 months, CDKN2A deletion, strongly correlates to a poor outcome in the entire cohort (5y OS=25% respectively vs.60% for patients in germline configuration, p=.003) and in the subgroup of patients treated by R-CHOP (5y OS=40% vs.70%, p=.04). Furthermore, prognosis impact of GCNA involving CDKN2A was validated in an independent set of 35 patients treated by R-CHOP. To conclude, combination of QMPSF and GEP may constitute a powerful approach to delineate new genomic pathways with prognosis impact in DLBCL. Notably, CDKN2A/CDKN2B loss, detected in more than one third of DLBCL patients constitutes a strong factor of chemoresistance that is not overcome by R combination. GEP indicates that this may be a consequence of an independent p14arf/p53 pathway, involving the well-established p14arf related ribosome regulation function.

Basic fibroblast growth factor impact on retinoblastoma progression and survival.

Chemotherapy resistance is a problem in the treatment of advanced retinoblastoma (RB). Since basic fibroblast growth factor (bFGF) is a survival factor for neural precursor cells, bFGF was evaluated as a growth and chemoresistance factor in RB. bFGF expression was analyzed in the LH-betaTag transgenic mouse model of RB and human RB cell lines by immunofluorescence, RT-PCR, and Western blot. Proliferation and apoptosis (TUNEL) assays were performed. bFGF levels significantly increased during tumorigenesis in transgenic RB, as a function of tumor status (P = 0.005). PCR and confocal microscopy confirmed that the human cell lines and primary tumors expressed bFGF. bFGF was localized to vascular and tumor cells and rarely to glial cells. Exogenous 18-kDa bFGF induced proliferation in two RB cell lines (WERI and Y79). Western blot analysis demonstrated 34-, 22-, and 18-kDa isoforms in transgenic RB and both cell lines. In TUNEL assays, chemoresistance to carboplatin-induced apoptosis was observed in the Y79 line, which expressed a higher ratio of high (34 kDa)- to low-molecular-weight bFGF isoforms, compared with the WERI line. Similar to other bFGF tumor studies, exogenous low-molecular-weight (18 kDa) bFGF (1 ng) significantly enhanced carboplatin-induced apoptosis in the more chemosensitive WERI, but not the chemoresistant Y79 line. RB tumors produce significant amounts of bFGF, and the differential production and response to isoforms of bFGF may have implications for invasive tumor growth and chemoresistance.

ShRNA-targeted Cyclooxygenase (COX)-2 inhibits proliferation, reduces invasion and enhances chemosensitivity in laryngeal carcinoma cells

Cyclooxygenase-2 (COX-2), one isoform of cyclooxygenase proinflammatary enzymes, is a causal factor for tumor development, invasion, metastasis, and chemoresistance. It is frequently overexpressed in a variety of human malignancies, including laryngeal carcinoma. To investigate its possibility as a therapeutic target for the treatment of laryngeal carcinoma, we employed RNA interference technology to downregulate endogenous gene COX-2 expression in laryngeal carcinoma cells and analyzed its phenotypical changes. Results showed that shRNA-mediated downregulation of COX-2 expression in human laryngeal carcinoma cells significantly inhibited cell proliferation and colony formation in vitro and reduced the potential of tumorigenicity in vivo. The specific downregulation led to cell arrest in the G(0)/G(1) phase of cell cycle and final apoptosis induction. The increased apoptosis was associated with the ratios of Bcl-2 or Bcl-xL/Bax. In the present study, we also observed that the downregulation of COX-2 could obviously enhanced the cytotoxic effect of Taxanes both in vitro and in vivo. All these results suggest that knockdown of COX-2 expression can lead to potent antitumor activity and chemosensitizing activity to taxanes in human laryngeal carcinomas.

O6- Methylguanine-DNA-Methyltransferase in Recurring Anaplastic Ependymomas: PCR and Immunohistochemistry

Ependymomas are the third most common brain tumor in children. The post surgical management is controversial. There are no convincing data on an effective role for chemotherapy. O6-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. Hypermethylation of the MGMT gene promoter is an important cause of MGMT inactivation. We evaluated the MGMT gene promoter methylation and the immunohistochemical MGMT protein expression in 12 recurrent anaplastic ependymomas affecting children. Our purpose was to investigate the molecular rationale of the administration of alkylating agents to children affected by recurrent anaplastic ependymomas. All ependymomas lacked MGMT promoter hypermethylation and 9 (75%) showed high MGMT protein expression (>50% tumoral cells). Differences between different recurrences in the same patient were not observed. These results may indicate MGMT as a factor of chemoresistance to alkylating drugs in anaplastic ependymomas and support the uncertainties regarding the actual benefit of chemotherapy for patients with anaplastic ependymomas.

Quantitative image analysis of intra-tumoral bFGF level as a molecular marker of paclitaxel resistance

BackgroundThe role of basic fibroblast growth factor (bFGF) in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The goal of the present study was to quantify bFGF levels in archived tumor tissues, and to determine its relationship with chemosensitivity.MethodsWe established an image analysis-based method to quantify and convert the immunostaining intensity of intra-tumor bFGF to concentrations; this was accomplished by generating standard curves using human xenograft tumors as the renewable tissue source for simultaneous image analysis and ELISA. The relationships between bFGF concentrations and tumor chemosensitivity of patient tumors (n = 87) to paclitaxel were evaluated using linear regression analysis.ResultsThe image analysis results were compared to our previous results obtained using a conventional, semi-quantitative visual scoring method. While both analyses indicated an inverse relationship between bFGF level and tumor sensitivity to paclitaxel, the image analysis method, by providing bFGF levels in individual tumors and therefore more data points (87 numerical values as opposed to four groups of staining intensities), further enabled the quantitative analysis of the relationship in subgroups of tumors with different pathobiological properties. The results show significant correlation between bFGF level and tumor sensitivity to the antiproliferation effect, but not the apoptotic effect, of paclitaxel. We further found stronger correlations of bFGF level and paclitaxel sensitivity in four tumor subgroups (high stage, positive p53 staining, negative aFGF staining, containing higher-than-median bFGF level), compared to all other groups. These findings suggest that the relationship between intra-tumoral bFGF level and paclitaxel sensitivity was context-dependent, which may explain the previous contradictory findings on the merit of using plasma or urine bFGF level as a prognostic indicator.ConclusionThe present study established a quantitative image analysis method that enabled the measurement of intratumoral bFGF level in archived tissues. The ability to quantify a potential biomarker provided the opportunity to study the relationship between the biomarker and chemosensitivity in tumor subgroups and thereby enabled hypothesis generation for additional translational research.

Expression of glutathione S-transferase T1 (GSTT1) in human brain tumours.

Glutathione S-transferases (GSTs) play a central role in a number of metabolic processes. Glutathione S-transferase T1 (GSTT1) is a polymorphic cytosolic enzyme and a member of the theta class of GSTs. Typical substrates for GSTT1 are industrial compounds, such as dichloromethane and ethylene oxide. It has been shown that also chemotherapeutic drugs such as BCNU [i.e. 1,3-bis(2-chloroethyl)-1-nitrosourea] are efficiently inactivated by GSTT1. BCNU is a drug which is increasingly used locally in the chemotherapy of glioblastoma multiforme WHO grade IV. Therefore, if GSTT1 were expressed in neoplastic cells of brain tumours it could be a factor for chemoresistance. In order to clarify a possible role of GSTT1 in chemoresistance, as a first step, we localized this enzyme in malignant gliomas such as glioblastoma multiforme WHO grade IV and oligodendroglioma WHO grade II. Because of its polymorphism we first genotyped the samples for GSTT1 by PCR. Using in situ hybridization, we then demonstrated that GSTT1 transcripts are expressed in neoplastic cells of both tumour types. Immunohistochemistry revealed then that whereas neoplastic cells in glioblastoma multiforme WHO grade IV contain GSTT1, it was not localized in oligodendroglioma cells. Given the polymorphism of GSTT1 and its potential activity towards BCNU, the localization of GSTT1 in glioblastoma cells can be considered as a possible factor of non-homogeneous chemotherapy response among patients with different GSTT1 genotypes.

GRP78 as a Novel Predictor of Responsiveness to Chemotherapy in Breast Cancer

The discovery of predictive factors for chemoresistance is critical for improving adjuvant therapy for cancer patients. The 78-kDa glucose-regulated protein (GRP78), widely used as an indicator of the unfolded protein response (UPR), is induced in the tumor microenvironment. In vitro studies suggest that GRP78 confers chemoresistance to topoisomerase inhibitors, such as Adriamycin (doxorubicin). Here, we report on a retrospective cohort study of 127 stage II and III breast cancer patients who were treated with Adriamycin-based chemotherapy. Archival tumor specimens were available for analysis and the relationship of GRP78 expression level to "time to recurrence" (TTR), used as a surrogate marker for drug resistance, was examined. Our data show that 67% of the study subjects expressed high level of GRP78 in their tumors before the initiation of chemotherapy and suggest an association between GRP78 positivity and shorter TTR [hazard ratio (HR), 1.78; P = 0.16]. Interestingly, subgroup analysis reveals that the HR for the GRP78-positive group increased significantly among patients who did not receive further taxane treatment (HR, 3.00; P = 0.022) and among mastectomy patients (HR, 3.33; P = 0.027). The HR was even stronger among mastectomy patients who did not receive further taxane treatment (HR, 4.82; P = 0.010). The use of GRP78 as a predictor for chemoresponsiveness and the potential interaction of GRP78 and/or the UPR pathways with taxanes warrant larger studies.

Expression of p53R2 Is Related to Prognosis in Patients with Esophageal Squamous Cell Carcinoma

The p53 gene and its family are important factors for carcinogenesis, prognosis, and chemoresistance in esophageal squamous cell carcinoma. A recently identified ribonucleotide reductase, p53R2, is regulated by p53 for supplying nucleotides to repair damaged DNA. In the present study, we analyzed the expression and clinicopathologic significance of p53 and p53R2 in esophageal squamous cell carcinoma. We immunohistochemically investigated the relationship between p53 and p53R2 expressions in surgical specimens of primary tumors in 222 patients with esophageal squamous cell carcinoma. The positive expression rate of p53 was 47.1% and that of p53R2 was 61.7%. Positive p53R2 expression was significantly correlated with depth of invasion, lymph node metastasis, stage, and poor prognosis. In the p53-negative group, the 5-year survival rate was better in patients with negative p53R2 expression than in those with positive p53R2 expression. Multivariate analysis indicated that the negative expression of both p53 and p53R2 was an independent prognostic factor along with tumor depth nodal metastasis and stage. We showed that positive p53R2 expression was related to tumor development and that alteration of p53R2 expression in p53-negative tumors was closely related to prognosis. Evaluation of the expressions of p53 and p53R2 proteins should be useful for determining the tumor properties, including prognosis, in patients with esophageal squamous cell carcinoma.

Prediction of Anticancer Drug Potency from Expression of Genes Involved in Growth Factor Signaling

This study develops and evaluates a systematic approach to finding biomarker genes for predicting potency of anticancer drugs against tumor cells, focusing on gene families related to growth factor signaling. Cytotoxic potencies of 119 drugs against 60 neoplastic cell lines (NCI-60) were correlated with expression of 343 genes, including 90 growth factors and receptors, 63 metalloproteinases, and 92 ras-like GTPases as downstream signaling factors. Progressively more stringent criteria and predictive models aim at identifying the smallest subset of genes predictive of cytotoxic potency. Comparing gene expression with drug potency across the NCI-60 yielded genes with negative and positive correlations (p < 0.001), indicative of a role in chemoresistance and chemosensitivity, respectively. Of 17 genes with multiple negative correlations, 8 are known chemoresistance factors, validating the approach. Negatively correlated genes clustered into two main groups with distinct expression profiles and drug correlations, represented by EGFR and ERBB2 (Her-2/Neu). Accordingly, no synergism was observed between EGFR and ERBB2 inhibitors. However, combinations with classical anticacer drugs were not correlated with EGFR and ERBB2 expression in four cell lines tested, suggesting complex interactions in combination treatments. Finally, a subset of only 13 genes was found to be sufficient for near optimal prediction of drug potency against the NCI-60. Our approach using a small subset of genes reveals known and potential biomarkers in cancer chemotherapy, providing a strategy for genome-wide analysis.

Bcl-XL depletion promotes apoptosis and retards growth of pancreatic cancer tumor xenografts

Predominant Bcl-XL overexpression in pancreatic cancer cells blocks apoptosis, and may factor in chemoresistance. Bcl-XL depletion reverses this, enhancing chemotherapy-induced caspase activation, and TNF-alpha/TRAIL-induced apoptosis in vitro. We examined whether Bcl-XL depletion would alter tumor growth in vivo. Bcl-XL knockdown RNAi vectors were designed, constructed and introduced by retroviral infection into Panc-1 pancreatic cancer cells. A separate group of Panc-1 cells were similarly infected using a control RNAi vector.