Chemoresistance Genes Research Articles

Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death

Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85±19.30days) in GBM but not in astrocytomas (59.13±47.26days) and oligodendrogliomas (58.04±59.80days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an attractive candidate for potentially overcoming therapy-resistance not only in malignant glioma but in broad spectrum of cancers.

Mithramycin A sensitizes therapy-resistant breast cancer stem cells toward genotoxic drug doxorubicin

Chemotherapy resistance is a major clinical challenge for the management of locally advanced breast cancer. Accumulating evidence suggests a major role of cancer stem cells (CSCs) in chemoresistance evoking the requirement of drugs that selectively target CSCs in combination with chemotherapy. Here, we report that mithramycin A, a known specificity protein (Sp)1 inhibitor, sensitizes breast CSCs (bCSCs) by perturbing the expression of drug efflux transporters, ATP-binding cassette sub-family G, member 2 (ABCG2) and ATP-binding cassette sub-family C, member 1 (ABCC1), survival factors, B-cell lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP), and, stemness regulators, octamer-binding transcription factor 4 (Oct4) and Nanog, which are inherently upregulated in these cells compared with the rest of the tumor population. In-depth analysis revealed that aberrant overexpression of Sp1 in bCSCs transcriptionally upregulates (1) resistance-promoting genes to protect these cells from genotoxic therapy, and (2) stemness regulators to sustain self-renewal potential of these cells. However, mithramycin A causes transcriptional suppression of these chemoresistant and self-renewal genes by inhibiting Sp1 recruitment to their promoters. Under such antisurvival microenvironment, chemotherapeutic agent doxorubicin induces apoptosis in bCSCs via DNA damage-induced reactive oxygen species generation. Cumulatively, our findings raise the possibility that mithramycin A might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to eliminate CSCs. This will consequently lead to the improvement of therapeutic outcome for the treatment-resistant breast carcinomas.

Abstract 4404: CSF-1R signaling drives mesothelioma stemness and chemoresistance

Abstract Clinical management of Malignant Pleural Mesothelioma (MPM) is very challenging because of the uncommon resistance of the tumor to chemotherapy. We started from the observation that 7/7 mesothelioma cell lines tested expressed both the Colony Stimulating Factor Receptor-1 and its -ligands CSF-1 and IL-34, a signaling module mainly characterized in hematopoietic cell subpopulations. Pemetrexed and cisplatin treatment did not affect the number of mesothelioma CSF1R-expressing cells. Additionally, interference with CSF1R expression or function significantly abrogated the resistance of these cells to pemetrexed, suggesting the involvement of CSF-1R signaling in mediating chemoresistance of mesothelioma. This prompted us to isolate and characterize the CSF-1Rpos cells and we found that the purified cells expressed a complex repertoire of pluripotency markers, EMT factors and chemoresistance genes. The functions of the CSF-1R expressing MPM cells relied on the autocrine production of CSF-1 and IL-34, whose downregulation affected the viability of the purified cells. We identified AKT, NFkB and STAT3 activation as distinct features of the CSF-1Rpos cells. We showed that all three pathways contributed to the survival of the CSF1Rpos cells, with AKT exerting the most relevant effects. The forced activation of CSF-1R in untransformed mesothelial cells was sufficient to confer clonogenicity and resistance to pemetrexed, typical feature of transformed mesothelial cells. Furthermore, this induced a gene expression profile very similar to that observed in the FACS sorted CSF-1Rpos cells, suggesting a causal relationship between CSF-1R activation and cell transformation. Active AKT signaling contributed to increased levels of nuclear β-catenin in the CSF-1R expressing cells. Inhibition of AKT reduced the transcriptional activity of β-catenin dependent reporters and sensitized the CSF-1Rpos cells to pemetrexed-induced senescence, thereby reversing the resistance of the CSF1Rpos cells to pemetrexed. This correlated with downregulation of c-MYC, a functional CSF-1R target, and upregulation of p21. Finally, we recapitulated the main findings in primary mesothelioma cultures, which exhibited long-term surviving, chemoresistant CSF-1Rpos cells of mesothelial nature and sensitive to CSF-1R inhibition. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, suggests that CSF-1R signaling may have a critical pathogenic role in malignant pleural mesothelioma and therefore may represent a promising target for therapeutic intervention. Citation Format: Mario Cioce, Claudia Canino, Chandra Goparaju, Haining Yang, Michele Carbone, Harvey I. Pass. CSF-1R signaling drives mesothelioma stemness and chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2014-4404

Abstract 5190: Detection of radioresistant gene of esophageal cancer by the new cyclopedic gene analysis with transposon

Abstract Backgound Radiation is one of the most important therapies same as operation and chemotherapy for esophageal cancer. However a rate of therapeutic effect is low, 68.3% as a chemoradiation therapy for advanced esophageal cancer in Japan. Predicting an effect before radiation therapy is impossible, many patients suffer from side effect of radiation without enough therapeutic effect. To predict and improve an effect, it is need to identify genes and mechanism of radioresistance. Aim of this research is to detect radioresistant gene of esophageal cancer by the new cyclopedic gene analysis with transposon. Methods Transposon is a base sequence which transports to other location in the genome at random. Inserting CMV promotor to transposon as a transcriptional activator, the following gene of transported transposon is overexpressed and the gene inserted transposon is knocked down. We can get various cells which have a different overexpressed or knocked down gene by transposon method. After drug treatment or irradiation for cells inserted transposon and CMV promotor, named transposon tagged cells, survived cells have overexpressed redioresistant genes or knocked down radiosensitive genes. We can pick up candidate redioresistant genes to detect the location of transposon in survived cells. We have already succeeded to acquire candidate chemoresistant genes of breast cancer and esophageal cancer. Results Twenty days after irradiation of 9 to 11 Gy for transposon tagged cells, survived cells became colonies. Picking up survived colonies and analyzing its gene, nine genes were detected as a candidate gene. All genes were knocked down by inserting the transposon. Knock down of candidate genes were demonstrated by decreasing an amount of mRNA in survived colonies. Cells of survived colonies also showed higher survival rate after irradiation than the rate of parent esophageal cancer cells without transposon. These results mean that knocking down of the candidate gene relates to redioresistance actually. Cytochrome c oxidase subunit 1 is one of the gene detected by transposon method. Cytochrome c oxidase 1 is known as an enzyme of Cytochrome c which induce apoptosis after DNA damage from radiation by activating caspase cascade. This fact supports that knock down of cytochrome c oxidase 1 may suppress release of cytochrome c and then radiation induced apoptosis. Conclusion Transposon method is able to analyze resistant genes comprehensively. To reveal resistant mechanism of detected gene, new target of treatment may be found. Citation Format: Tomoko Takesue. Detection of radioresistant gene of esophageal cancer by the new cyclopedic gene analysis with transposon. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2014-5190

Reversion of trichostatin A resistance via inhibition of the Wnt signaling pathway in human pancreatic cancer cells.

Drug resistance is a major impediment to successful chemotherapy in pancreatic cancer(PC) patients. We investigated the effect of Wnt/β-catenin signaling inhibition by wnt-c59 on chemoresistance in a trichostatin A-resistant Panc-1 cell line (Panc-1/TSA). Panc-1/TSA cells were treated with the Wnt/β‑catenin signaling inhibitor wnt-c59 (10µmol · l-1) and/or trichostatin A (TSA; 10µmol · l-1) for 24h. CCK-8 assay was utilized to analyze the interactive effect of TSA and wnt-c59 on induction of apoptosis of the Panc-1/TSA cells. Cell apoptosis was measured by flow cytometry. Real-time PCR and western blotting were used to assess Wnt/β-catenin signaling, epithelial-mesenchymal transition(EMT) and multidrug resistance (MDR). Real-time cell analysis(RTCA) was used to detect the cell migration ability. After wnt-c59 treatment for 24h, relative genes and transcriptional targets of Wnt/β-catenin signaling were downregulated (P<0.05). CCK-8 assay indicated that the combination of TSA and wnt-c59 had a synergistic effect on induction of Panc-1/TSA cell apoptosis. As detected by FACS, cell apoptosis rates increased significantly (P<0.05). The results of RTCA showed that the cell indices of the control group, wnt-c59 group, TSA group and TSA+wnt-c59 combination group were 1.2842±0.0257, 1.2155±0.0282, 1.2533±0.0194 and 0.8541±0.0250, respectively. In accordance, MMP-9 protein in the wnt-c59 treatment groups was decreased compared to the non-wnt-c59 treatment groups. Meanwhile, E-cadherin protein was upregulated and vimentin protein was downregulated, both of which are characteristic markers of EMT. Chemoresistant gene MDR1 and P-glycoprotein (P-gp) in the wnt-c59 treatment groups had a reduced expression compared to the non-wnt-c59 treatment groups. This study revealed that TSA sensitivity, migration ability, and the EMT phenotype in Panc-1/TSA cells were reversed following Wnt/β-catenin signaling inhibition.

EIF5A2 is a novel chemoresistance gene in breast cancer

The eIF5A2 gene (encoding the eukaryotic initiation factor 5A2) located at 3q26 is a putative oncogene that is overexpressed in colon and rectal carcinomas, lung cancer and hepatocellular carcinoma. EIF5A2 overexpression correlates significantly with tumor metastasis and is an adverse prognostic marker. However, eIF-5A2 overexpression in breast cancer and its effect on chemotherapy are unknown. We measured eIF-5A2 expression and doxorubicin sensitivity in different human breast cancer cell lines (Bcap-1937, HCC1937, and MCF-7). To investigate a role for eIF-5A2 in chemoresistance, cells were treated with eIF-5A2-siRNA, exposed to various concentrations of doxorubicin, and toxicity was assayed by CCK-8 (cell counting kit). The eIF-5A2 expression levels varied among breast cancer cells. Higher expression levels correlated with decreased doxorubicin sensitivity. Silencing of eIF-5A2 significantly improved doxorubicin toxicity in all three breast cancer cell lines. This study shows that eIF-5A2 plays an important role in doxorubicin chemoresistance in breast cancer cells.

S100P enhances the chemosensitivity of human gastric cancer cell lines

The effect of the protein S100P on biological characteristics of cancer is not clear, especially in gastric cancer. We previously showed that S100P positive gastric cancer patients have a better cumulative survival than S100P negative patients. To study the possible mechanisms of S100P enhanced the chemosensitivity to oxaliplatin in gastric cancer cell lines. S100P was overexpressed in vitro by plasmid transfection and downregulated by siRNA transfection in the BGC823 and SGC7901 gastric cancer cell lines. Cell survival rate, changes in the chemoresistance gene, such as GST-π, MDR1, MRP1, Topo-II, MVP and BCRP, intake of anticancer drug were measured after oxaliplatin treatment. In SGC7901 cells, MTT assay indicated that increased S100P expression levels decreased the survival rate and decreased S100P expression levels increased the survival rate. In BGC823 and SGC7901 cell lines, mRNA of MDR1, a chemoresistance genes, was decreased in cells that overexpressed S100P, and increased in cells with downregulation of S100P. Intracellular accumulation of platinum increased in cells with overexpressed S100P, and decreased in cells with S100P downregulation. S100P contributes to oxaliplatin chemosensitivity in gastric cell lines by increasing drug inflow. It might also be a novel independent prognostic factor in gastric cancer patients who receive adjuvant chemotherapy with oxaliplatin.

Activation of the nuclear receptor FXR enhances hepatocyte chemoprotection and liver tumor chemoresistance against genotoxic compounds

The success of pharmacological treatments in primary liver cancers is limited by the marked efficacy of mechanisms of chemoresistance already present in hepatocytes. The role of the nuclear receptor FXR is unclear. Although, in non-treated liver tumors, its expression is reduced, the refractoriness to anticancer drugs is high. Moreover, the treatment with cisplatin up-regulates FXR. The aim of this study was to investigate whether FXR is involved in stimulating chemoprotection/chemoresistance in healthy and tumor liver cells. In human hepatocytes, the activation of FXR with the agonist GW4064 resulted in a significant protection against cisplatin-induced toxicity. In human hepatoma Alexander cells, with negligible endogenous expression of FXR, GW4064 also protected against cisplatin-induced toxicity, but only if they were previously transfected with FXR/RXR. Investigation of 109 genes potentially involved in chemoresistance revealed that only ABCB4, TCEA2, CCL14, CCL15 and KRT13 were up-regulated by FXR activation both in human hepatocytes and FXR/RXR-expressing hepatoma cells. In both models, cisplatin, even in the absence of FXR agonists, such as bile acids and GW4064, was able to up-regulate FXR targets genes, which was due to FXR-mediated trans-activation of response elements in the promoter region. FXR-dependent chemoprotection was also efficient against other DNA-damaging compounds, such as doxorubicin, mitomycin C and potassium dichromate, but not against non-genotoxic drugs, such as colchicine, paclitaxel, acetaminophen, artesunate and sorafenib. In conclusion, ligand-dependent and independent activation of FXR stimulates mechanisms able to enhance the chemoprotection of hepatocytes against genotoxic compounds and to reduce the response of liver tumor cells to certain pharmacological treatments.

Genomics of T-ALL Reveals a Weapon of an Evasive Foe

Genomic profiling of T-ALL samples at diagnosis, remission, and relapse identifies NT5C2 as a chemoresistance gene.

Regulation of Excision Repair Cross-Complementation Group 1 by Snail Contributes to Cisplatin Resistance in Head and Neck Cancer

We investigated the mechanism and clinical significance of the epithelial-mesenchymal transition (EMT)-induced chemoresistance in head and neck squamous cell carcinoma (HNSCC). The correlation between the expression of different EMT regulators and chemoresistance genes, such as excision repair cross complementation group 1 (ERCC1), was evaluated in cancer cell lines from the NCI-60 database and four human HNSCC cell lines. Ectopic expression of Snail or short-interference RNA-mediated repression of Snail or ERCC1 was done in HNSCC cell lines. Cell viability was examined for cells after cisplatin treatment. A luciferase reporter assay and chromatin immunoprecipitation were used to identify the transcriptional regulation of ERCC1 by Snail. Immunohistochemical analysis of Snail, Twist1, ERCC1, hypoxia inducible factor-1 α (HIF-1α), and NBS1 were done in samples from 72 HNSCC patients receiving cisplatin-based chemotherapy. The correlation between the expression of Snail and ERCC1 was confirmed in different cell lines, including HNSCC cells. In HNSCC cell lines, overexpression of Snail in the low endogenous Snail/ERCC1 cell lines FaDu or CAL-27 increased ERCC1 expression, and hypoxia or overexpression of NBS1 also upregulated ERCC1. Knockdown of Snail in the high endogenous Snail/ERCC1 cell line OECM-1 downregulated ERCC1 expression and attenuated cisplatin resistance. Furthermore, suppression of ERCC1 in Snail- or NBS1-overexpressing HNSCC cells enhanced sensitivity to cisplatin. Snail directly regulated ERCC1 transcription. In patients with HNSCC, coexpression of Snail and ERCC1 correlated with cisplatin resistance and a poor prognosis. Activation of ERCC1 by Snail is critical in the generation of cisplatin resistance of HNSCC cells.

Expression of Chemoresistance-Related Genes and Heat Shock Protein 72 in Hyperthermic Isolated Limb Perfusion of Malignant Melanoma: An Experimental Study

Hyperthermic isolated limb perfusion (HILP) is considered an established treatment for multiple locoregional intransit metastases in malignant melanoma of the extremities. Various mechanisms such as the expression of chemoresistance genes and heat shock proteins by the tumor may be responsible for varying response rates and locoregional recurrences of the treatment. The aim of the experimental animal study was to investigate the direct impact of HILP on such mechanisms of resistance. Tissue temperature, administration of the cytostatic drug, and duration of perfusion were varied. Expression of the chemoresistance genes mdr1, mrp1, mrp2, and lrp and of heat shock protein 72 (HSP72) in the tumor tissue was analysed using RT-PCR and western blot analysis. The untreated SK-MEL-3 tumor expressed mdr1, mrp1, and lrp, but not mrp2. Neither variation of temperature, administration of the cytostatic drug, nor duration of perfusion changed the expression of this “resistance pattern”. In contrast to the cytostatic drug, hyperthermia causes a persistent induction of HSP72. Both observations could offer a potential explanation for failure of HILP in malignant melanoma.

APRIL is a novel clinical chemo-resistance biomarker in colorectal adenocarcinoma identified by gene expression profiling

Background5-Fluorouracil(5FU) and oral analogues, such as capecitabine, remain one of the most useful agents for the treatment of colorectal adenocarcinoma. Low toxicity and convenience of administration facilitate use, however clinical resistance is a major limitation. Investigation has failed to fully explain the molecular mechanisms of resistance and no clinically useful predictive biomarkers for 5FU resistance have been identified. We investigated the molecular mechanisms of clinical 5FU resistance in colorectal adenocarcinoma patients in a prospective biomarker discovery project utilising gene expression profiling. The aim was to identify novel 5FU resistance mechanisms and qualify these as candidate biomarkers and therapeutic targets.MethodsPutative treatment specific gene expression changes were identified in a transcriptomics study of rectal adenocarcinomas, biopsied and profiled before and after pre-operative short-course radiotherapy or 5FU based chemo-radiotherapy, using microarrays. Tumour from untreated controls at diagnosis and resection identified treatment-independent gene expression changes. Candidate 5FU chemo-resistant genes were identified by comparison of gene expression data sets from these clinical specimens with gene expression signatures from our previous studies of colorectal cancer cell lines, where parental and daughter lines resistant to 5FU were compared. A colorectal adenocarcinoma tissue microarray (n = 234, resected tumours) was used as an independent set to qualify candidates thus identified.ResultsAPRIL/TNFSF13 mRNA was significantly upregulated following 5FU based concurrent chemo-radiotherapy and in 5FU resistant colorectal adenocarcinoma cell lines but not in radiotherapy alone treated colorectal adenocarcinomas. Consistent withAPRIL's known function as an autocrine or paracrine secreted molecule, stromal but not tumour cell protein expression by immunohistochemistry was correlated with poor prognosis (p = 0.019) in the independent set. Stratified analysis revealed that protein expression of APRIL in the tumour stroma is associated with survival in adjuvant 5FU treated patients only (n = 103, p < 0.001), and is independently predictive of lack of clinical benefit from adjuvant 5FU [HR 6.25 (95%CI 1.48-26.32), p = 0.013].ConclusionsA combined investigative model, analysing the transcriptional response in clinical tumour specimens and cancers cell lines, has identified APRIL, a novel chemo-resistance biomarker with independent predictive impact in 5FU-treated CRC patients, that may represent a target for novel therapeutics.

Upregulation of antioxidants and chemoresistance genes in human granulosa cells following Pregnane X receptor activation: a potential role for fertility preservation

OBJECTIVE: The nuclear Pregnane X receptor (PXR; NR1I2) is an important component of the body's adaptive defense mechanism against toxic substances including foreign chemicals (xenobiotics). PXR stimulate CYP3A4 and MDR1 gene expression in the mouse liver. Here we investigate the expression of chemoresistance (PXR and MDR1) and antioxidants (CYP3A4 and NOQ1) genes in mural and granulosa cells from patients undergoing IVF as well as in human ovarian tissues.

Direct evidence that heterogeneity necessitates and limits the use of multidrug chemotherapy in colon cancer

Considerable progress in the form of multidrug chemotherapy has recently been made in chemotherapy for the prolongation of survival in advanced colon cancer. It is generally accepted that colon cancer is biologically heterogeneous for multiple properties, including sensitivity to chemotherapeutic agents and metastasis. Although this partly explains the success of multidrug chemotherapy, there has been no direct evidence that multidrug regimens affect individual heterogenous cancer characteristics in colon cancer. Here, we present a case of metachronous ovarian metastasis in a colon cancer patient with dissemination who underwent irinotecan-based followed by oxaliplatin-based chemotherapy. We were able to obtain three samples from the patient, one of primary cancer and two of metastatic tumors from secondary surgery. Of note, both chemoresistant and chemosensitive tumors were present in the patient at the same time. To understand the influence of multidrugs on individual cancer characteristics, we examined differences in the molecular characteristics of the three samples using RT-PCR, focusing in particular on alterations in chemoresistant genes. In shrunken peritoneal metastasis, we found a significant increase in the mRNA levels of an irinotecan-sensitive gene, although other molecular factors were resistant to both 5-FU and oxaliplatin. We also confirmed that the recurrent ovarian tumor showed significant resistance to all three drugs: 5-FU, irinotecan and oxaliplatin. These results suggest that the heterogeneity of colon cancer necessitates and limits the use of multidrug chemotherapy.

Real-time polymerase chain reaction technique determines absolute copy number of plasma methylated MGMT gene promoter copies in newly diagnosed malignant glioma patients

22073 Background: New methods for disease surveillance, quantification, and prognosis in malignant glioma are needed. Recent work has demonstrated that malignant gliomas release their DNA into the blood which bears the genetic and epigenetic aberrations in the intracranial lesion. We determined the actual plasma copy number of the methylated chemoresistance gene methylguanine methyltransferase (MGMT) in patients enrolled in clinical trials for malignant glioma as a prelude to biomarker development. Methods: This prospective NABTT CNS Consortium study was designed to determine if tumor specific plasma DNA could serve as an accurate biomarker. Plasma was collected from newly diagnosed high grade glioma patients prior to therapy and with each follow-up MRI. DNA was isolated from plasma of the first 19 patients enrolled and bisulfite treated. Quantitative PCR was performed with methylation-sensitive primers selective for the methylated MGMT promoter. A S. cerevisiae actin gene spike-in quantitative PCR technique was used to control for variations in DNA loss during the process. Resuts: Baseline plasma from 19 patients demonstrated an overall mean of 2.74 × 105 copies/mL (SEM: 7.67 × 104) of the MGMT promoter. The range of tumor specific DNA in these samples was 1.14 × 104 to 1.22 × 106 copies/mL. One of 19 patients demonstrated no detectable MGMT methylation. Conclusions: Tumor DNA in the blood of high grade glioma patients offers a unique platform to develop a biomarker to satisfy the needs of evolving treatments. This provides the possibility to assess tumor burden in a manner distinct from MRI scans which rely heavily on blood-brain barrier status. This study will eventually yield important information on initial tumor specific DNA concentrations in plasma and changes over the course of the patients' illness. No significant financial relationships to disclose.

Alteration of the chemoresistant gene expression during chemotherapy for colon cancer: A molecular case report

Chemotherapy remains the main choice of treatment for the management of unresectable or metastatic colorectal cancer. However, drug resistance is a major problem, limiting the effectiveness of the chemotherapies presently used to treat cancer. During treatment, drug resistance can also be acquired by tumors that are initially sensitive to chemotherapy. We present a case of metachronous splenic recurrence after a curative resection of appendical cancer with ovarian metastasis, although the patient had been treated with 5-FU/LV followed by mFOLFOX6 after surgery. Molecular analyses by RT-PCR also showed that the residual tumor after chemotherapy has cancer cells overexpressing 5-FU/l-OHP based chemotherapy-resistant genes. Therefore, it was suggested that a careful assessment of the disease status be undertaken during chemotherapy to ensure that the possibility of surgical resection, especially of the re-growth or partial response tumors, was not missed, since several genes, chemoresistant to the agents used, can be induced in residual tumors during chemotherapy.

Evaluation of the MDR1, ABCG2, Topoisomerases IIα and GSTπ gene expression in patients affected by aggressive mantle cell lymphoma treated by the R-Hyper-CVAD regimen

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIα, glutathione-s-transferaseπ (GSTπ) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTπ and topoisomerase IIα. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.

Colony-Stimulating Factor-1 Antibody Reverses Chemoresistance in Human MCF-7 Breast Cancer Xenografts

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringer's solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.

The chemoresistance gene ABCG2 (MXR/BCRP1/ABCP1) is not expressed in melanomas but in single neuroendocrine carcinomas of the skin

As the molecular mechanisms in melanoma chemoresistance remain unknown to date, we hypothesized these tumors to express the ATP-binding cassette (ABC) transporter G2 (ABCG2/MXR/BCRP1/ABCP1), a recently detected membrane transporter and putative stem-cell marker. Besides melanoma, we addressed the neuroendocrine carcinoma of the skin (Merkel cell carcinoma), another cutaneous cancer supposed to originate from neuroectoderm and usually chemoresistant. Upon semiquantitative reverse transcription polymerase chain reaction, ABCG2 mRNA expression was not upregulated in 18 melanoma resection specimens when compared with 19 acquired melanocytic nevi from which melanomas are known to often arise (Mantel-Haenszel test, p=0.3). At protein level, immunohistochemistry was negative in all 66 investigated melanoma resection specimens (50 primary melanomas and 16 cutaneous/subcutaneous metastases) and in 19 acquired melanocytic nevi. Among 29 neuroendocrine carcinomas of the skin, ABCG2 protein was detected in single clusters of cells in three tumors. As a positive control, three dermatofibrosarcomas were also stained and showed ABCG2 protein expression of the endothelial cells of the blood vessels. Altogether, chemoresistance of melanomas and neuroendocrine carcinomas of the skin cannot be explained by expression of the ABCG2-chemoresistance gene. Most of these tumors do not exhibit this potential stem-cell feature.

Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.