Chemotherapy-resistant Phenotype Research Articles

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance.

Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

Toll-like receptor 2 promotes breast cancer progression and resistance to chemotherapy

ABSTRACT Cancer stem cells (CSCs) are the main drivers of disease progression and chemotherapy resistance in breast cancer. Tumor progression and chemoresistance might then be prevented by CSC-targeted therapies. We previously demonstrated that Toll-like Receptor (TLR)2 is overexpressed in CSCs and fuels their self-renewal. Here, we show that high TLR2 expression is linked to poor prognosis in breast cancer patients, therefore representing a candidate target for breast cancer treatment. By using a novel mammary cancer-prone TLR2KO mouse model, we demonstrate that TLR2 is required for CSC pool maintenance and for regulatory T cell induction. Accordingly, cancer-prone TLR2KO mice display delayed tumor onset and increased survival. Transplantation of TLR2WT and TLR2KO cancer cells in either TLR2WT or TLR2KO hosts shows that tumor initiation is mostly sustained by TLR2 expression in cancer cells. TLR2 host deficiency partially impairs cancer cell growth, implying a pro-tumorigenic effect of TLR2 expression in immune cells. Finally, we demonstrate that doxorubicin-induced release of HMGB1 activates TLR2 signaling in cancer cells, leading to a chemotherapy-resistant phenotype. Unprecedented use of TLR2 inhibitors in vivo reduces tumor growth and potentiates doxorubicin efficacy with no negative impact on the host immune system, opening new perspectives for the treatment of breast cancer patients.

Chemotherapy-elicited exosomal miR-378a-3p and miR-378d to promote breast cancer stemness and chemoresistance via the activation of EZH2/STAT3 signaling.

e12615 Background: Not all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemo-elicited exosomes in regulating chemoresistance is poorly understood. Methods: Using commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs. Results: Here, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome induced drug resistance in a nude mouse tumor xenograft model. Conclusions: This study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

Chemotherapy-elicited exosomal miR-378a-3p and miR-378d promote breast cancer stemness and chemoresistance via the activation of EZH2/STAT3 signaling

BackgroundNot all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemotherapy-elicited exosomes in regulating chemoresistance is poorly understood.MethodsUsing commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs.ResultsHere, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome-induced drug resistance in a nude mouse tumor xenograft model.ConclusionThis study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of the WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

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ROR1 Expression and Its Functional Significance in Hepatocellular Carcinoma Cells.

Background: Hepatocellular carcinoma (HCC) is a common and deadly cancer; however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. Methods: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. Results: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. Conclusions: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

P08.58 Impact of selected pentabromobenzylisothiourea (ZKK-3) combined with hyperbaric oxygen therapy on therapeutic response and oxygenation status of T98G glioblastoma cell line

Abstract Introduction: Glioblastoma (GBM), the primary brain tumour derived from astroglial cells, is the most frequently diagnosed intracranial malignancy in adults. Poor prognosis for GBM patients results from high invasiveness of neoplastic cells related with faulty angiogenesis and persistent hypoxia states. In such conditions glioma cells develop radio- and chemotherapy-resistant phenotype. It is postulated that application of hyperbaric oxygen (HBO) may improve therapeutic response via refinement of tumour tissue oxygenation. The more effective therapeutic strategies are also required. One of the novel compounds that exhibit anti-tumour properties are pentabromobenzylisothioureas. The aim of this study was to examine whether HBO may enhance anti-tumour efficacy of selected pentabromobenzylisothiourea when both agents were administered as combined ZKK-3/HBO therapy. Materials and Methods: Human glioblastoma T98G cell line was cultured in medium supplemented with novel isothiourea derivative - ZKK-3. The proliferation and viability of glioma cells cultured in hypoxia or hyperbaric oxygen conditions were examined. Cell proliferation was tested 24 hours after ZKK-3 addition using Nikon Inverted Microscope and Multisizer 3 Beckman Coulter. The viability assay was taken 24 and 48 hours post ZKK-3 supplementation by CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega). Hypoxia state of T98G cells was determined via the evaluation of HIF-1α protein expression using HIF-1A ELISA Kit (Thermo Scientific). Tests were performed on cells treated with ZKK-3 and cultured in various oxygen conditions: 1/ normoxia (24 hours), 2/ hypoxia (24 hours), 3/ HBO (2 ATA, 1 hour followed by 23 hours of normoxia), 4/ double hypoxia, 5/ hypoxia/HBO. Results: Proliferation of T98G cells treated with ZKK-3 was significantly decreased after hyperbaric oxygenation in comparison to hypoxia conditions. Administration of HBO resulted also in statistically significant diminution of glioma cells’ viability. Expression of HIF-1α in T98G cells strongly depended on oxygen conditions: in hypoxia and double hypoxia the level of protein was significantly elevated compared with normoxia, while after hyperbaric oxygenation no changes or even decrease of HIF-1α level was observed. Comparison of double hypoxia and hypoxia/HBO groups showed that HBO addition after ZKK-3 supplementation caused significant reduction of HIF-1α expression. Conclusions: Treatment of malignant glioma cells with selected isothiourea derivative is significantly more efficient when combined with hyperbaric oxygen administration. Beneficial effect of HBO on decreasing HIF-1α expression allows to consider hyperbaric oxygenation as the way to overcome tumour hypoxia.ACKNOWLEDGEMENT: The research was supported by the KNOW-MMRC project and Foundation for the Development of Diagnostic and Therapy.

Abstract 1558: The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis

Abstract Introduction: Metastatic disease is responsible for 90% of colorectal cancer (CRC) deaths. Studies suggest that metastasis is closely associated with the presence of CRC tumor-initiating cells (Co-TIC) and their interaction with the lymph node (LN) stromal microenvironment. Prior to developing extra-nodal metastasis, these cells acquire a chemotherapy-resistant phenotype developing genetic alterations making them resistant to conventional treatments. In addition to cell-cell contact and secreted molecules, a recently discovered means of intercellular signaling is the exchange of extra-cellular vesicles. These microvesicles (MVs) carry complex biological information, including mRNA, miRNA, as well as soluble and transmembrane proteins that can affect the behavior of target cells. MVs have been detected in patient specimens with diverse malignancies and may play a role in communication between the LN stromal microenvironment and Co-TIC. We hypothesize that MVs are involved in intracellular trafficking between LN stromal cells and CRC cells promoting tumor formation and distant organ metastasis. Methods: MVs released by human mesenteric LN stromal cells (LNSC) derived from surgical specimens and the established LN stromal cell line (HK cell) were isolated using differential centrifugation and gradient purification. The MVs were visualized using GFP-HK cell and RFP-HT-29 cell (CRC cell line) and florescence microscopy. The functional properties of LN stromal MVs and their effect on CRC proliferation and metastasis was analyzed using established in vitro co-culture models and a humanized orthotopic intra-rectal (IR) injection mouse model, tracked by bioluminescent imaging (BLI). Results: A 100,000 g pellet containing MVs derived from LNSC and HK cells have a similar size profile when analyzed by NanoSight. Budding CD63-RFP tagged MVs were released by LNSC and HK cells and uptake by GFP tagged CRC cells was confirmed through time-lapse experiments using deconvoluting microscopy. When HK cell or LN stromal cell-derived MVs were co-cultured with HT-29 cells in vitro, they supported HT-29 cell growth at a similar level as that of HK cell or LN stromal cell conditioned media, respectively. By adding LNSC- or HK-derived MVs to HT-29-Luc cells or patient derived CRC cells (CRC-Pt-Luc cells) in our IR model, we demonstrated that MVs enhanced CRC tumor growth as well as distant organ metastasis in vivo. Conclusion: MVs isolated from LNSCs traffic between the stromal cells and CRC cells. These MVs promote tumor formation and distant organ metastasis in vivo suggesting that they play a crucial role in the communication between the LN stromal microenvironment and CRC cells. Further analyzing the functional properties of effector MV RNAs may help identify novel targetable candidates for therapeutic strategies that target CRC metastasis using our unique patient derived orthotopic mouse model. Citation Format: Xin Zhang, Ryan Sullivan, Nathan Hite, Grace Maresh, Linh Hellmers, Zhen Lin, Erik Flemington, Carlos Salomon, Heather Green, David Margolin, Li Li. The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis. [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 1558.

The potential of clofarabine in MLL-rearranged infant acute lymphoblastic leukaemia

MLL-rearranged acute lymphoblastic leukaemia (ALL) in infants is the most difficult-to-treat type of childhood ALL, displaying a chemotherapy-resistant phenotype, and unique histone modifications, gene expression signatures and DNA methylation patterns. MLL-rearranged infant ALL responds remarkably well to nucleoside analogue drugs in vitro, such as cytarabine and cladribine, and to the demethylating agents decitabine and zebularine as measured by cytotoxicity assays. These observations led to the inclusion of cytarabine into the treatment regimens currently used for infants with ALL. However, survival chances for infants with MLL-rearranged ALL do still not exceed 30–40%.Here we explored the in vitro potential of the novel nucleoside analogue clofarabine for MLL-rearranged infant ALL. Therefore we used both cell line models as well as primary patient cells. Compared with other nucleoside analogues, clofarabine effectively targeted primary MLL-rearranged infant ALL cells at the lowest concentrations, with median LC50 values of ∼25nM. Interestingly, clofarabine displayed synergistic cytotoxic effects in combination with cytarabine. Furthermore, at concentrations of 5–10nM clofarabine induced demethylation of the promoter region of the tumour suppressor gene FHIT (Fragile Histidine Triad), a gene typically hypermethylated in MLL-rearranged ALL. Demethylation of the FHIT promoter region was accompanied by subtle re-expression of this gene both at the mRNA and protein level. We conclude that clofarabine is an interesting candidate for further studies in MLL-rearranged ALL in infants.

MiR-205 impairs the autophagic flux and enhances cisplatin cytotoxicity in castration-resistant prostate cancer cells

Compelling evidence suggests that epithelial-to-mesenchymal transition is involved in the resistance of human cancer cells to chemotherapy. We previously reported that the expression of miR-205, a miRNA down-regulated in prostate cancer, is further repressed in prostate cancer cells undergoing epithelial-to-mesenchymal transition, suggesting a possible involvement of the miRNA in the acquisition of the chemoresistant phenotype. In the present study, we show that miR-205 replacement in castration-resistant mesenchymal prostate cancer cells caused an enhancement of cisplatin cytotoxic activity in vitro and in vivo, as a consequence of autophagy impairment. Specifically, the constraints on the autophagic flux were associated to the miRNA-dependent down-regulation of the lysosome-associated proteins RAB27A and LAMP3. These findings suggest that miR-205-mediated impairment of the autophagic pathway may interfere with the detoxifying capabilities of prostate cancer cells in their attempt to cope with cisplatin-induced detrimental effects. Overall, our data indicate that (i) loss of miR-205 may indeed contribute to acquire mesenchymal tracts and concomitantly establish a permissive autophagic milieu that confers a chemotherapy resistant phenotype to prostate cancer cells, and (ii) strategies aimed at restoring miR-205 expression levels may represent a successful approach to overcome resistance of prostate cancer to platinum compounds.

Abstract 323: High grade serous ovarian cancer cells spontaneously self-organize in vitro into multicellular irregular aggregates and highly organized spheroids.

Abstract Ovarian cancer (OvCa) has the worst prognosis among gynecologic cancers because it is typically detected at an advanced stage. Accumulation of fluid inside the peritoneal cavity, and multicellular clusters featured within ascites are common manifestations of the disease. Patients undergo aggressive cytoreductive surgery followed by platinum-taxane therapy; yet in most cases, patients will relapse and die due to tumor progression. OvCa recurs as it presents, with growths within the peritoneal cavity, accumulation of ascites, but with a chemotherapy-resistant phenotype. Thus, understanding the dynamics of peritoneal metastases is of clear significance. This work provides evidences that OvCa cells derived from serous, treatment-resistant relapsed disease spontaneously develop multicellular anchorage-independent OvCa aggregates in vitro. The human serous ovarian adenocarcinoma PEO6 cells, while growing as adherent monolayer, spontaneously self-assemble into multicellular foci that float away off the adherent component of the culture. The floating phenotype is comprised of irregular cellular clusters and more organized spheroids with blastocyst-like morphology. Within each multicellular structure cells gather differently as evidenced by the distribution pattern of the cell junction adhesion molecule E-cadherin. Both types of non-adherent multicellular aggregates have live cells with no morphological features of apoptosis indicating their resistance to anoikis. Anchorage independent OvCa spheroids have the capacity to recreate the entire culture phenotype when placed in a new dish; the spheroids adhere upon transference and develop foci that grow upright irregular aggregates and more organized spheroids, ultimately leading to the generation of new non-adherent components. The floating PEO6 structures include clusters of cells expressing the cancer initiating antigen CD133 supporting their plasticity to perpetuate the anchorage-independent component of the culture. In summary, this study led to the characterization of an in vitro model of recurrent, treatment-resistant serous OvCa cells with intrinsic ability to gather in two types of clusters that survive in an anchorage-independent manner-a hallmark of transformation and critical factor in metastasis. The spontaneous in vitro development of OvCa multicellular irregular aggregates and spheroids at recurrence may represent a preclinical model to study the biology of selection and adaptation of disease malignancy and ways to interrupt its progression. Citation Format: Alicia A. Goyeneche. High grade serous ovarian cancer cells spontaneously self-organize in vitro into multicellular irregular aggregates and highly organized spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2013-323

Genetically engineered natural killer (NK) cell immunotherapy for children poor risk b-cell (CD20+) leukemia and lymphoma (L/L).

e13022 Background: A majority of children with CD20+ L/L at relapse have a chemotherapy resistant phenotype (Cairo et al Blood, 2007; JCO, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this poor risk population. NK cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). PBNK cells expanded with genetically engineered K562-mbIL15-41BBL cells (geK562) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of anti-CD20 chimeric antigen receptor (CAR+) engineered PBNK cells expanded with mK562 against CD20+ L/L both in vitro and in vivo. Methods: Peripheral blood mononuclear cells (PBMC) were expanded with mitomycin C treated geK562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. Retrovirus preps that express CAR+ or CAR- were generated independently. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD). Expanded PBMC were transduced with retroviruses as described (Imai C et al, Blood. 2005). NK cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3- PBNK cells were significantly increased compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, p<0.001). CD56-CD3+ PBT cells were significantly reduced compared to media alone at day 7 (22.08+2.22% vs 75.73+0.75%, n=6, p<0.001). CAR+ and CAR- retrovirus supernants infected expanded PBMC at 1%-10% range. The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with CAR+ PBNK compared to CAR- PBNK (41+ 1.1% vs 24.5+ 3.7%) against Ramos at E:T ratio 2:1. Conclusions: PBNK can be expanded with geK562. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include characterizing the cytotoxicity activity of engineered PBNK against L/L in vitro and survival in xenogafted mice.

Abstract 3511: Genetically engineered natural killer (NK) cell immunotherapy for poor risk B-cell (CD20+) leukemia and lymphoma (L/L)

Abstract Background: Patients with CD20+ L/L at relapse have a chemotherapy resistant phenotype and a dismal prognosis (Cairo et al, Blood, 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Ex vivo expansion of PBNK cells with genetically engineered K562-mbIL15-41BBL cells (modified K562) and the expanded PBNK cells transduced with chimeric anti-CD19 antigen receptor (CAR) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of chimeric anti-CD20 antigen receptor (CAR+) engineered PBNK cells expanded with K562-mbIL15-41BBL cells (modified K562) against CD20+ L/L both in vitro and in vivo. Methods: Whole peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. PBMC were incubated with mitomycin C modified K562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD, St. Jude Children's Hospital). CAR+ and CAR− retroviruses were generated in 293T cells with helper plasmids. Jurkat cells were used to examine infection efficiency. Anti-CD20 CAR expression was examined by flow cytometry and by western blot of whole cell lysates for CD3zeta. PBMC stimulated with modified K562 were transduced with retroviruses as previous described (Imai C et al, Blood. 2005). PBNK cells cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3− PBNK cells were expanded significantly more following modified K562 stimulation compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, P<0.001). CD56−CD3+ PBT cells were significantly reduced compared to media alone at day7 (22.08+2.22% vs 75.73+0.75%, n=6, P<0.001). CAR+ and CAR− retrovirus supernants infected Jurkat cells at 40%-97% efficiency and infected expanded PBMC at 1%-10% range (n=3). The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with anti-CD20 CAR+ PBNK compared to CAR− PBNK (41+ 1.1% vs 24.5+ 3.7%, n=3) against Ramos at effector to target ratio 2:1. Conclusion and Future Directions: PBNK can be significantly induced following modified K562 stimulation. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include further characterizing the cytotoxicity activity of Anti-CD20 CAR engineered expanded PBNK cells against L/L in-vitro and survival in xenogafted mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3511. doi:1538-7445.AM2012-3511

Genetically Engineered Natural Killer (NK) Cell Immunotherapy for Poor Risk B-Cell (CD20+) Leukemia and Lymphoma

Abstract 1003 Background:Patients with CD20+ Leukemia and Lymphoma (L/L) at diagnosis and a majority of those at relapse have a chemotherapy resistant phenotype and a dismal prognosis. Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells from cord blood (CB) play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically Engineered expanded CBNK cells with anti-CD19 chimeric receptor has been previously reported (Li L et al, Cancer Gene Ther. 2010). Objective:We investigated the functional activities and cytolytic effect of chimeric anti-CD20 receptor (anti-CD20 CAR) engineered CBNK cells expanded with modified K562 (generously supplied by D. Camapana, MD, PhD) against CD20+ L/L both in vitro and in vivo. Methods:CBMNC were obtained by Ficoll-Hypaque density gradient centrifugation. CBMNC were cocultured with 100Gy irradiated MODK562 (K562-mbIL15-41BBL cells) or wildtype (WT) K562 in RPMI+ 10 IU/ml IL-2 for 7 days. Medium was changed every 2 days with fresh medium and IL-2. CD56 and CD3 expression were evaluated by flow cytometry. NK activation marker LAMP-1 and cytolytic mechanism markers, perforin and granzyme B were evaluated by flow cytometry in expanded CBNK. NK cytotoxicity was assessed by europium release assay at 20:1 E:T ratio against CD20+ Ramos. Luciferase expressing (ffLuc+) Ramos were generated by transfecting ffLUCZeo-pcDNA plasmid DNA (generously supplied by L. Cooper, MD, PhD) and selected by Zeocin. In vivo ffLuc+ Ramos engraftment in NOD/SCID mice was verified using a Xenogen IVIS-200 system (Caliper Life Sciences). 6 weeks old xenografted mice were treated for 5 weeks with PBS, Ramos, 5×106 WTK562 expanded (E) CBMNC, 5×106 or 5×107 MODK562E CBMNC IP. Tumor growth was monitored by volume using bioluminescent imaging and mice survival was observed for 10 weeks. Different retrovirus preps that express the anti-CD20 CAR or control GFP only (CAR−) were generated independently. The anti-CD20 CAR was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD, St. Jude Children’s Hospital, Memphis, Tenn). MSCV-anti-CD20BB-CD3-zeta-GFP or control empty vector (MSCV-GFP) were transfected to 293T cells with helper plasmids pEQ-PAM3(-E) and pRDF. Conditioned medium containing retrovirus was harvested 48 and 72 h after transfection. Jurkat cells were used to examine infection efficiency. Anti-CD20 CAR expression was examined by flow cytometry with staining cells with goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin (Jackson Immunoresearch, West Grove, PA, USA) followed by PE-conjugated streptavidin and the expression was further confirmed by western blot of whole cell lysates for CD3zeta. Results:CBNK cells were significantly increased compared to WTK562 and media alone (72 ± 4 {35-fold} vs 43 ± 6 vs 9 ± 2%, p<0.01) (Figure 1). Cytotoxicity against Ramos was increased compared to WTK562 CBMNC (p<0.01); along with increased expression of NK activation marker, CD107a (p<0.05), perforin and granzymeB (42 ± 1.5 vs 15 ± 0.5%,p<0.001; 22 ± 0.5 vs 11 ± 0.3%, p<0.0.001, respectively). At 5 weeks, tumor volume in mice receiving either dose of MODK562E CBMNC was significantly decreased vs WTK562E CBMNC (2 ± 0.6 and 0.4 ± .05 vs 3 ± 0.25/mm3, p=0.0086, p=0.001, respectively and luminescence p<0.01). Xenografted mice receiving MODK562E CBMNC showed increased survival vs WTK562E (p<0.001). CAR+ and CAR− retrovirus supernants infected Jurkat cells efficiently. 3 independent retrovirus preps infected Jurkat cells with 97.71% CAR−GFP+ vs 80.25% CAR+ GFP+, 40.25% CAR−GFP+ vs 74.03% CAR+ GFP+, and 88.57% CAR−GFP+ vs 62.64% CAR+ GFP+. The anti-CD20 CAR expression was further confirmed by western blots (Figure 2). Conclusion and Future Directions:CBMNC stimulated with MODK562 was associated with significantly increased NK LAMP-1, perforin and granzyme B expression, enhanced cytotoxicity in-vitro and in-vivo survival. Anti-CD20 CAR expressed was confirmed in infected cells. Future directions include infecting MODK562 expanded CBNK with retrovirus expressing anti-CD20 CAR and examining the cytotoxicity activity of engineered CBNK against L/L in-vitro and survival in xenogafted mice. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hedgehog signaling maintains chemoresistance in myeloid leukemic cells

The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. There is still limited insight into the molecular mechanisms that maintain the chemotherapy-resistant phenotype, despite the obvious clinical relevance that such knowledge would have. In this study, we show that the chemotherapy-resistant phenotype of myeloid leukemia cells correlates with activation of the Hedgehog (Hh) pathway, whereas in chemosensitive cells, such activation is less pronounced. Importantly, the overexpression of Hh pathway components induces chemoprotection and inhibition of the pathway reverts chemoresistance of Lucena-1 cells, apparently by interfering with P-glycoprotein-dependent drug resistance. Our data thus identify the Hh pathway as an essential component of multidrug resistance (MDR) myeloid leukemia and suggest that targeting the Hh pathway might be an interesting therapeutic avenue for overcoming MDR resistance in myeloid leukemia.

The actin-bundling motility protein fascin and vascular endothelial growth factor (VEGF) are universally over-expressed in human angiosarcoma

10068 Background: Angiosarcoma is a highly aggressive soft tissue malignancy with a dismal progression-free survival (PFS). Treatment options for disseminated disease are limited and present systemic therapies have not shown evidence of prolonging overall survival. In this regard, a greater understanding of the molecular and cellular basis of the tumor phenotype of this uncommon malignancy and how these features correlate to clinical behavior would be an important step towards identifying potential new molecular targets for therapy. Our previous work show that acquisition of the chemotherapy-resistant phenotype by angiosarcoma cells occurs concomitantly with a marked increase in cell motility and VEGF expression. Increased expression of the angiogenic factor VEGF and the motility protein fascin have been associated with more aggressive clinical course in a number of other tumor types and may play a direct role in the biologic behavior of these malignancies. Therefore, in the current study, we sought to characterize angiosarcoma by immunohistochemistry for the expression of VEGF and fascin, and compared the staining pattern to benign vascular tumors. Methods: 20 cases of angiosarcoma, and 5 cases of hemangioma were stained for tumor cell expression of VEGF and fascin using anti-VEGF polyclonal antibody (clone A-20, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA) and antibody to fascin (clone 55K-2, 1:500, DakoCytomation, Carpinteria, CA). Results: Surprisingly, every case of angiosarcoma showed high expression for both VEGF and fascin with moderate to strong staining intensity in 75 to 100% of the tumor cells for each case. By comparison, both proteins were either absent or only weakly positive in hemangiomas. Conclusions: In the current study we found high levels of VEGF and fascin universally expressed in angiosarcomas, which was not the case for benign vascular tumors. These factors maybe part of the malignant phenotype for these tumors and contribute to an aggressive clinical course through enhanced cell motility and autocrine/paracrine signaling. Additional study will be needed to better define a role for these proteins in angiosarcoma and to determine if they might represent potential new molecular targets for therapy. No significant financial relationships to disclose.

Tbx3 Is a Downstream Target of the Wnt/β-Catenin Pathway and a Critical Mediator of β-Catenin Survival Functions in Liver Cancer

Tbx3 encodes a transcriptional repressor that is important for diverse patterning events during development, and Tbx3 mutation in humans causes the ulnar-mammary syndrome. Here, we describe the identification of Tbx3 in array-based search for genes downstream Wnt/beta-catenin that are implicated in liver tumorigenesis. Overexpression of Tbx3 is closely associated with the mutational status of beta-catenin in murine liver tumors induced by Myc as well as in human hepatocellular carcinomas and hepatoblastomas. Moreover, Tbx3 transcription is activated by ectopic expression of beta-catenin in mouse liver and in human tumor cell lines. Evidence that Tbx3 transcription is directly regulated by beta-catenin is provided by chromatin immunoprecipitation and reporter assays. Although HepG2 cells stably transfected with Tbx3 display moderately enhanced growth rate, the dominant negative mutant Tbx3-Y149S drastically inhibits hepatoma cell growth in vitro and in vivo. Moreover, small interfering RNAs (siRNA) directed against Tbx3 inhibit anchorage-independent growth of liver and colon carcinoma cells. We further show that inhibition of Tbx3 expression by specific siRNAs blocks beta-catenin-mediated cell survival and renders cells sensitive to doxorubicin-induced apoptosis. Conversely, ectopic expression of Tbx3 inhibits apoptosis induced by beta-catenin depletion. Marked overexpression of Tbx3 in a subset of hepatoblastomas is associated with chemotherapy-resistant phenotype and unfavorable patient outcome. These results reveal an unsuspected role of Tbx3 as a mediator of beta-catenin activities on cell proliferation and survival and as an important player in liver tumorigenesis.

Can the state of cancer chemotherapy resistance be reverted by epigenetic therapy?

BackgroundTranscriptome analysis shows that the chemotherapy innate resistance state of tumors is characterized by: poorly dividing tumor cells; an increased DNA repair; an increased drug efflux potential by ABC-transporters; and a dysfunctional ECM. Because chemotherapy resistance involves multiple genes, epigenetic-mediated changes could be the main force responsible of this phenotype. Our hypothesis deals with the potential role of epigenetic therapy for affecting the chemotherapy resistant phenotype of malignant tumors.Presentation of the hypothesisRecent studies reveal the involvement of DNA methylation and histone modifications in the reprogramming of the genome of mammalian cells in cancer. In this sense, it can be hypothesized that epigenetic reprogramming can participate in the establishment of an epigenetic mark associated with the chemotherapy resistant phenotype. If this were correct, then it could be expected that agents targeting DNA methylation and histone deacetylation would by reverting the epigenetic mark induce a global expression profile that mirror the observed in untreated resistant cells.Testing the hypothesisIt is proposed to perform a detailed analysis using all the available databases where the gene expression of primary tumors was analyzed and data correlated with the therapeutic outcome to determine whether a transcriptome profiling of "resistance" is observed. Assuming an epigenetic programming determines at some level the intrinsic resistant phenotype, then a similar pattern of gene expression dictated by an epigenetic mark should also be found in cell acquiring drug resistance. If these expectations are meet, then it should be further investigated at the genomic level whether these phenotypes are associated to certain patterns of DNA methylation and chromatin modification. Once confirmed the existence of an epigenetic mark associated to either the intrinsic or acquired chemotherapy resistant phenotype, then a causal association should be investigated. These preclinical findings should also be tested in a clinical setting.Implications of the hypothesisOur hypothesis on the ability of epigenetic therapy to revert the epigenetic changes leading to a transcritome profile that defines the resistant state will eventually be a more rational and effective way to treat malignant tumors.

An Expression Signature Classifies Chemotherapy-Resistant Pediatric Osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFbeta1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor-driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.