Primary Human Tumor Specimens Research Articles

MYC and HSF1 Cooperate to Drive PLK1 inhibitor Sensitivity in High Grade Serous Ovarian Cancer.

Ovarian cancer is a deadly female cancer with high rates of recurrence. The primary treatment strategy for patients is platinum-based therapy regimens that almost universally develop resistance. Consequently, new therapeutic avenues are needed to overcome the plateau that current therapies have on patient outcomes. We describe a gene amplification involving both HSF1 and MYC, wherein these two genes on chromosome 8q are co-amplified in over 7% of human tumors that is enriched to over 30% of patients with ovarian cancer. We further found that HSF1 and MYC transcriptional activity is correlated in human tumors and ovarian cancer cell lines, suggesting they may cooperate in ovarian cancer cells. CUT&RUN for HSF1 and MYC in co-amplified ovarian cancer cells revealed that HSF1 and MYC have overlapping binding at a substantial number of locations throughout the genome where their binding peaks are near identical. Consistent with these data, a protein-protein interaction between HSF1 and MYC was detected in ovarian cancer cells, implying these two transcription factors have a molecular cooperation. Further supporting their cooperation, growth of HSF1-MYC co-amplified ovarian cancer cells were found to be dependent on both HSF1 and MYC. In an attempt to identify a therapeutic target that could take advantage of this dependency on both HSF1 and MYC, PLK1 was identified as being correlated with HSF1 and MYC in primary human tumor specimens, consistent with a previously established effect of PLK1 on HSF1 and MYC protein levels. Targeting PLK1 with the compound volasertib (BI-6727) revealed a greater than 200-fold increased potency of volasertib in HSF1-MYC co-amplified ovarian cancer cells compared to ovarian cancer cells wild-type HSF1 and MYC copy number, which extended to several growth assays, including spheroid growth. Volasertib, and other PLK1 inhibitors, have not shown great success in clinical trials and this study suggests that targeting PLK1 may be viable in a precision medicine approach using HSF1-MYC co-amplification as a biomarker for response.

A Mechanism Linking Id2-TGFβ Crosstalk to Reversible Adaptive Plasticity in Neuroblastoma

The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing, anoikis-resistant anchorage independent (AI) phenotypes. Both phenotypes are present in established mouse and human tumors. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt, Erk, Bcl2 and integrins was observed in AI cells. Similarly, differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines, SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in 50% cure of microscopic disease and delayed tumor growth in established mouse neuroblastoma tumors. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma.

Abstract A240: A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma.

Abstract The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with a remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing anoikis resistant anchorage independent (AI) phenotypes. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt, Erk, Bcl2, and integrins was observed in AI cells. Similarly, differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines, SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/ Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in cure of established tumors in a mouse model of neuroblastoma. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A240. Citation Format: Lina Chakrabarti, Bi-Dar Wang, Norman H. Lee, Anthony Sandler. A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A240.

Loss of ATM increases pancreatic cancer formation via induction of EMT

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths in the developed world, with a 5 year survival rate as low as 5%. In recent years, genetically engineered animal models of pancreatic cancer, which recapitulate the human disease, have provided useful means of studying both the morphological and molecular features associated with PDAC. The most commonly studied model to date has been the expression of active Kras under the control of an endogenous, pancreas specific promoter, which leads to a spectrum of changes in the pancreas recapitulating development of human PDAC. Interestingly, PDAC develops much more rapidly when further oncogenes such as SMAD4 or p53 are deleted. In the current study, we address the role of Ataxia Telangiectasia Mutated (ATM) gene in PDAC and describe a loss of ATM protein with advanced UICC stage in primary human tumor specimens. We also found that mutations in the ATM gene are more abundant in large collectives of human pancreatic cancer. Subsequently, we show that loss of ATM enhanced the metastatic potential of the Kras mouse model and decreased survival of mice to 36 and 38 weeks for homozygous and heterozygous ATM deletion, respectively. These mice developed a greater number of pre-malignant pancreatic intraepithelial neoplasias (mPanINs) with a significant increase in proliferation and α-smooth muscle actin positive cells, indicative of activated pancreatic stellate cells (PSCs). This was followed by an increase in large, dysplastic papillary lesions and a significant amount of surrounding fibrosis compared to controls. Our results furthermore establish that the loss of ATM in this mouse model promoted EMT via an increased response to BMP4-signalling. Moreover, early EMT upon ATM loss was associated with increased invasive properties. Taken together, our data suggests that there may be an intimate link between ATM activity and pancreatic cancer formation, one of the major signalling pathways altered in PDAC and targeting this axis may provide therapeutic potential in the future.

Reversible adaptive plasticity: a mechanism for neuroblastoma cell heterogeneity and chemo-resistance.

We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered, anchorage dependent (AD) or sphere forming, anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin, self-renewal capacity, and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2, β-catenin, and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice, tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity, respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

Profound Coordinated Alterations of Intratumoral NK Cell Phenotype and Function in Lung Carcinoma

Both the innate and adaptive immune systems contribute to tumor immunosurveillance in mice and humans; however, there is a paucity of direct evidence of a role for natural killer (NK) cells in this important process. In this study, we investigated the intratumoral phenotypic profile and functions of NK cells in primary human tumor specimens of non-small cell lung carcinoma (NSCLC). We used in situ methods to quantify and localize NK cells using the NKp46 marker and we characterized their phenotype in blood, tumoral, and nontumoral samples of NSCLC patients. Intratumoral NK cells displayed a profound and coordinated alteration of their phenotype, with a drastic reduction of NK cell receptor expression specifically detected in the tumoral region. According to their altered phenotype, intratumoral NK cells exhibited profound defects in the ability to activate degranulation and IFN-γ production. We found that the presence of NK cells did not impact the clinical outcome of patients with NSCLC. Finally, we showed that tumor cells heterogeneously express ligands for both activating and inhibitory NK receptors. Taken together, our results suggest that the NSCLC tumor microenvironment locally impairs NK cells, rendering them less tumorcidal and thereby supportive to cancer progression.

Abstract 5000: The functional stem cell marker aldehyde dehydrogenase enhances pancreatic cancer stem cell isolation and correlates with clinical prognosis

Abstract Pancreatic adenocarcinoma is a highly aggressive cancer that is largely resistant to current therapies and portends a median survival for patients of 9-12 months. Resistance may be due to a subpopulation of cells that are chemoresistant and able to support tumor propagation, such as cancer stem cells (CSCs). CSCs have emerged as attractive therapeutic targets, but the development of effective therapies requires the isolation of highly purified cell populations. Surface antigen expression has been used to identify CSC in most systems, however, methods based on functional properties, such as Hoechst dye exclusion or aldehyde dehydrogenase (ALDH) activity, can enhance stem cell isolation from normal and malignant tissues. We compared CSCs from pancreatic adenocarcinomas isolated by cell surface phenotype, ALDH activity, or both. Initially, we studied ALDH expression in human pancreatic cancer cell lines (CAPAN-1, DAN-G, CFPAC-1) using the Aldefluor assay. We found that each line contained a small population of ALDH+ cells (0.9-6.5% of total cells) that demonstrated increased clonogenic growth potential both in vitro and in vivo compared to ALDH- cells. Next, we compared ALDH expression with the published pancreatic CSC phenotype (CD24+CD44+) in cell lines and low-passage xenografts derived from primary human tumor specimens. Interestingly, we found that the putative CSC populations were only partially overlapping since 7-18.2% of the ALDH+ cells were CD44+CD24+ and only 4.2-36.2% of the CD44+CD24+ cells expressed ALDH. We compared CD44+CD24+ and CD44+CD24+ALDH+ cells isolated from 5 unique human pancreatic cancer xenografts and found that the CD44+CD24+ALDH+ cells were more tumorgenic in NOD/SCID mice. We also evaluated ALDH expression in primary pathologic specimen from 268 patients with pancreatic cancer by immunohistochemistry and found small, intensely positive cells with round or oval nuclei near the base of cancerous glands or at the invasive front in 90 (34%) of those patients. Furthermore, the presence of these cells was significantly associated with poorer overall survival (14 vs. 18 mos, p=0.02). We have begun to evaluate the mechanism by which pancreatic CSCs may be involved in more aggressive disease and found that CD44+CD24+ALDH+ cells isolated from tumor xenografts have significantly lower expression of E-cadherin and higher expression of Slug, a transcriptional repressor of E-cadherin, compared to ALDH+ or ALDH- cells. Therefore, ALDH appears to mark highly clonogenic pancreatic CSCs. Moreover, increased features associated with epithelial-to-mesenchymal transition may play a role in the correlation of ALDH+ cells with long-term outcomes.

Optimization of a metastasizing human gastric cancer model in nude mice.

Our purpose was to optimize the surgical orthotopic implantation (SOI) technique to create a reproducible gastric cancer model in nude mice with stable tumor growth and metastasizing course. We performed xenotransplantation of primary human tumor specimens from patients with gastric cancer (series 1) and orthotopic transplantation of tumor specimens originating from the gastric cancer cell line 23132/87 (series 2). All specimens were transplanted using microsurgical techniques. The two series were compared with regard to tumor growth rates and kinetics, development of metastases, and induction of minimal residual disease (MRD), as determined by histology and PCR techniques. In series 1 mice, the tumor growth rate was slow; in series 2 mice, it was both fast and reproducible. Unlike animals in series 1, animals in series 2 developed metastases and MRD. In conclusion, the optimized SOI technique presented here represents a reproducible and reliably metastasizing gastric cancer model.

19. Conditionally Replicative Adenoviruses Expressing p53 or Targeted towards EGFR Exhibit Enhanced Oncolytic Potency on Primary Human Tumor Specimens In Vitro and In Vivo

19. Conditionally Replicative Adenoviruses Expressing p53 or Targeted towards EGFR Exhibit Enhanced Oncolytic Potency on Primary Human Tumor Specimens In Vitro and In Vivo

Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished.

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and alpha(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

Activity of SCH 66336, a tricyclic farnesyltransferase inhibitor, against human tumor colony-forming units

The ras gene product regulates transduction of growth-proliferative signals from the membrane to the nucleus. Mutationally-activated Ras is the oncogene most frequently found in human tumors. In order to perform its function in cell signaling, Ras must be farnesylated on the CAAX motif present on the carboxyl terminus of the ras protein. This reaction is catalysed by farnesyl protein transferase. In the present study, SCH 66336, an orally bioavailable nonpeptide tricyclic farnesyltransferase inhibitor, was tested against a large variety of human tumors to define its preclinical activity profile, utilizing the human tumor cloning assay. A soft agar cloning assay was used to determine the in vitro effects of SCH 66336 against primary human tumor specimens taken directly from patients. A total of 70 evaluable specimens were exposed to SCH 66336 for 14-day continuous exposure at concentrations ranging from 0.1 to 2.5 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. There was a positive relationship between concentration and response to SCH 66336. With the highest concentration (2.5 microM), response was demonstrated in 50% (three of six) of breast tumors, 40% (6 of 15) of ovarian tumors, and 38% (5 of 13) of non-small-cell lung tumor colony forming units. Among the 69 specimens tested at the concentration of 2.5 microM, SCH 66336 had activity in 27% of tumor specimens that were resistant to doxorubicin, 38% of tumor specimens resistant to cisplatin, 33% of tumor specimens resistant to paclitaxel, and 27% of tumor specimens resistant to etoposide. The broad spectrum of soft agar growth inhibition by SCH 66336 in the human tumor cloning assay, and its efficacy at physiologically relevant concentrations in animal models, suggest that SCH 66336 may deserve future clinical trials in patients with ovarian, breast and non-small-cell lung cancer.

In vitro antitumor activity of the novel marine agent, Ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients

Ecteinascidin-743 (ET-743), a member of the ecteinascidin family selected for clinical development, is a tetrahydroisoquinolone alkaloid isolated from the marine ascidian, Ecteinascidia turbinata. This novel compound is a minor groove binding, guanine-specific alkylating agent which also interacts with the microtubule network and blocks cell cycle progression at late S/G2. A soft agar cloning assay was used to determine the in vitro effects of ET-743 against primary human tumor specimens taken directly from patients. A total of 93 evaluable specimens were exposed to ET-743 for one-hour (n = 25) and/or 14-day continuous exposure (n = 92) at concentrations ranging from 0.1 nM to 1 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. One-hour exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM induced in vitro responses in 0% (0/17), 6% (1/17), 16% (4/25), 13% (1/8), and 25% (2/8) of specimens, respectively. Continuous exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM, inhibited 0% (0/16), 13% (2/16), 49% (44/90), 62% (47/76), and 77% (58/75) of tumor specimens, respectively. Tumor-specific responses and concentration-dependent relationships were observed with a continuous exposure to ET-743. At 100 nM, the compound inhibited 79% (11/14) breast, 69% (9/13) non-small-cell lung, 58% (7/12) ovary, and 88% (7/8) melanoma specimens. At 1 microM, ET-743 inhibited 100% (14/14) breast specimens, 85% (11/13) non-small-cell lung, 67% (8/12) ovary and 86% (6/7) melanoma specimens. Activity of ET-743 at and above 10 nM was also observed against sarcoma and kidney tumors. At 10 nM concentration and continuous exposure ET-743 demonstrated incomplete cross-resistance with paclitaxel, alkylating agents, doxorubicin and cisplatin. Our data from the cloning assay indicate that the duration of exposure to ET-743 is an important factor in human tumors. Therefore, long-term exposure to ET-743 may be preferred in future clinical trials. The activity of ET-743 in breast, non-small-cell lung, and ovarian cancers as well as in melanoma may deserve further clinical evaluations. The potential of ET-743 in sarcoma and renal tumors might also be considered. In addition, our data indicate that a plasma concentration of 100 nM of ET-743 must be considered as a target during the clinical development of the compound; also the concept of continuous/protracted exposure in clinical trials with ET-743 has to be taken into account.

Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes.

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.

Effects of Recombinant Human Erythropoietin on Clonogenic Growth of Primary Human Tumor Specimens in vitro

Background: Production of Erythropoietin has been reported for various tumor cell lines in vitro. Also, a number of clinical reports indicate aberrant synthesis and release of erythropoietin, particularly in renal cell cancer patients. These findings raise the question whether erythropoietin, besides its hematopoietic lineage specificity, can exert an autocrine growth modulting effect on non-hematopoietic cells. Material and Methods: We have studied the effects of recombinant human erythropoietin (rhE) on in vitro clonogenic growth of human primary tumor specimens using a soft agar cloning system. Final concentrations ranged from 0.4-400 U/ml. Results: 53/116 (45.7%) tumor specimens were evaluable. Median colony formation in control capillaries without erythropoietin was 10.0 (range 3.0-274.0). Significant stimulation of clonal growth was observed in only two specimens (3.8%) and was not concentration-dependent. Five of 53 (9%) specimens showed inhibition of colony formation in at least one concentration of recombinant human erythropoietin. In four of these specimens, inhibition was observed at 400 U/ml only. Diluent controls were without effect. There was no evidence for a functional interaction between rhE and Interleukin (IL)-lΑ IL-lΒ, IL-3, IL-6, GM-CSF, or G-CSF.¹ This work was supported in part by grant W41/88/Ha 1 from the Deutsche Krebshilfe and a grant from the Wissenschaftliches Herausgeberkollegium der MÜnchener Medizinischen Wochenschrift.