Drive Tumor Formation Research Articles

Targeted p16(Ink4a) epimutation causes tumorigenesis and reduces survival in mice.

Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aberrant change in epigenetic regulation) can induce tumorigenesis. Here, we exploited a functionally validated cis-acting regulatory element and devised a strategy to induce developmentally regulated genomic targeting of DNA methylation. We used this system to target DNA methylation within the p16(Ink4a) promoter in mice in vivo. Engineered p16(Ink4a) promoter hypermethylation led to transcriptional suppression in somatic tissues during aging and increased the incidence of spontaneous cancers in these mice. Further, mice carrying a germline p16(Ink4a) mutation in one allele and a somatic epimutation in the other had accelerated tumor onset and substantially shortened tumor-free survival. Taken together, these results provide direct functional evidence that p16(Ink4a) epimutation drives tumor formation and malignant progression and validate a targeted methylation approach to epigenetic engineering.

The phosphatidylinositol 3-kinase (PI3K) isoform dependence of tumor formation is determined by the genetic mode of PI3K pathway activation rather than by tissue type.

Previous work has shown that prostate cancer in a Pten-null murine model is dependent on the p110β isoform of phosphatidylinositol 3-kinase (PI3K), while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110α dependent. Whether these differences in isoform dependence arise from tissue specificity or from the nature of the oncogenic signal activating the PI3K pathway is important, given increasing interest in using isoform-specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT-driven prostate cancer model was actually dependent on PI3K. A newly generated transgenic prostate line expressing an MT allele (Y315F) known to be defective for PI3K binding displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110α or p110β in mice in which wild-type MT was expressed in the prostate. We found that tumor formation driven by MT was significantly delayed by the loss of p110α expression, while ablation of p110β had no effect. Since the tumor formation driven by MT is p110α dependent in the prostate as well as in the mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type. Middle T antigen (MT), the oncogene of polyomavirus, can drive tumor formation in a variety of cell types and tissues. Interestingly, MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3K, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110α and p110β. Previous work in animal models showed that p110α was the key isoform in breast tumors driven by oncogenes, including MT and HER2, while p110β was key in prostate tumors driven by Pten loss. We asked the simple question of whether a prostate tumor driven by MT depends on p110α, which would suggest that the mode of activation determines p110 isoform dependence, or p110β, which would suggest that tissue type determines isoform dependence. The clear answer is that MT depends on p110α in both the prostate and breast.

Rescuing cell polarity with selective small molecule inhibitors of de‐palmitoylation (597.2)

Protein palmitoylation is a unique post‐translational modification required for the membrane anchoring and trafficking of numerous regulatory proteins that play key roles in cell growth, polarity, and signaling. In pulse‐chase palmitoylation proteomics studies, we identified the tumor suppressor Scrib as the most dynamic, enzymatically regulated palmitoylated protein in malignant cells. Scrib localizes at the basolateral membrane where it regulates epithelial cell apical‐basolateral polarity, junctional integrity, proliferation, and metastasis. Loss of Scrib at the plasma membrane cooperates with oncogenic Ras or Myc overexpression to drive tumor formation, promote epidermal to mesenchymal transition (EMT), and bypass contact inhibition. Interestingly, overexpression of Snail in polarized cells eliminates Scrib membrane localization. We recently reported a class of in vivo active small selective inhibitors of the acyl protein thioesterases APT1 and APT2. Addition of a small molecule APT2 inhibitor restores Scrib membrane localization and enhances E‐Cadherin expression, overcoming Snail‐mediated repression. These findings suggest that APT2, and not APT1, regulate the palmitoylation of cell polarity proteins, and highlight a strategy to attenuate oncogenic signaling and restore cell polarity pathways in malignant cells.Grant Funding Source: NIH R00CA151460

CD44v6 Is a Marker of Constitutive and Reprogrammed Cancer Stem Cells Driving Colon Cancer Metastasis

Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/β-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

Wnt Antagonist, Secreted Frizzled-Related Protein 4 (sFRP4), Increases Chemotherapeutic Response of Glioma Stem-Like Cells

Malignant gliomas have a highly tumorigenic subpopulation, termed cancer stem cells (CSCs), that drives tumor formation and proliferation. CSCs possess inherent resistance mechanisms against radiation- and chemotherapy-induced cancer cell death, enabling them to survive and initiate tumor recurrence. We examined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt signaling antagonist, in chemosensitizing the glioma cell line U138MG and glioma stem cells (GSCs) enriched from U138MG to chemotherapeutics. We found that sFRP4 alone and in combination with either doxorubicin or cisplatin induced apoptosis. Proliferation decreased substantially in GSC-enriched population as measured by MTT and BrdU assays. JC-1 and caspase-3 assays demonstrated that cell death was through the apoptotic pathway. sFRP4 treatment also decreased neurosphere formation and induced neuronal differentiation. Inhibition by sFRP4 was abolished by Wnt3a, indicating that sFRP4 acts through the frizzled receptor. Further indication that sFRP4 acts through the Wnt β-catenin pathway was provided by decrease in the β-catenin protein and decrease in the β-catenin-stimulated gene cyclin D1 upon sFRP4 induction. By real-time PCR, an increase in apoptotic markers Bax and p21, a decrease in pro-proliferative marker CycD1, and a decrease in the GSC marker CD133 were observed. These observations indicate that sFRP4 is able to sensitize glioma cells and stem cells to chemotherapeutics. We thus identified for the first time that sFRP4 could help to destroy cancer stem cells of glioma cell line, which would lead to effective treatment regimen to combat brain tumors.

Lung cancer stem cells, p53 mutations and MDM2.

Over the past few decades, advances in cancer research have enabled us to understand the different mechanisms that contribute to the aberrant proliferation of normal cells into abnormal cells that result in tumors. In the pursuit to find cures, researchers have primarily focused on various molecular level changes that are unique to cancerous cells. In humans, about 50 % or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Despite the identification of numerous triggers that causes lung cancer specific cure still remain elusive. One of the primary reasons attributed to this is due to the fact that the tumor tissue is heterogeneous and contains numerous sub-populations of cells. Studies have shown that a specific sub-population of cells termed as cancer stem cells (CSCs) drive the recurrence of cancer in response to standard chemotherapy. These CSCs are mutated cells with core properties similar to those of adult stem cells. They reside in a microenvironment within the tumor tissue that supports their growth and make them less susceptible to drug treatment. These cells possess properties of symmetric self-renewal and migration thus driving tumor formation and metastasis. Therefore, research specifically targeting these cells has gained prominence towards developing new therapeutic agents against cancer. This chapter focuses on lung cancer stem cells, p53 mutations noted in these cells, and importance of MDM2 interactions. Further, research approaches for better understanding of molecular mechanisms that drive CSC function and developing appropriate therapies are discussed.

Tracking down the origin of cancer: metabolic reprogramming as a driver of stemness and tumorigenesis.

Metabolic reprogramming has recently emerged as a fundamental trait of cancer cells. Initially thought to be a consequence of rapid cell proliferation, recent data have reset this idea by demonstrating that metabolic reprogramming can actually drive tumorigenesis. The cancer stem cell (CSC) theory predicts that only a small subpopulation of cancer cells with stem cell properties, which derive from the cancer cell of origin, possesses tumor-initiating potential. However, whether metabolic reprogramming drives tumor formation by regulating the genesis of CSCs is not known. Importantly, the metabolic properties of stem cells and cancer cells are strikingly similar, and metabolic reprogramming is a key factor controlling stemness in these cells. This article reviews the current understanding of cancer metabolism and how it mirrors the metabolic requirements of stem cells. These two concepts are integrated and data demonstrating that metabolic reprogramming regulates CSCs function are discussed, suggesting that metabolic regulation of stemness could be at the origin of cancer.

Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.

Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility.

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

Deletion of Irf5 protects hematopoietic stem cells from DNA damage-induced apoptosis and suppresses γ-irradiation-induced thymic lymphomagenesis

Repeated low-dose γ-irradiation (IR) induces thymic lymphoma in mice because of oncogenic mutations propagating from a primitive hematopoietic stem/progenitor cell (HSC) in the bone marrow. It is well known that IR-induced thymic lymphomagenesis is markedly enhanced by p53 deficiency, yet data also indicate that p53-dependent apoptosis can actively drive tumor formation in this model. The latter was recently expounded on by findings from Puma-deficient mice, indicating that loss of this proapoptotic p53 target gene results in protection from IR-induced lymphomagenesis rather than enhanced susceptibility to. Similar to Puma, the transcription factor interferon regulatory factor 5 (Irf5) has been reported as a p53 target gene and is required for DNA damage-induced apoptosis. To date, no studies have been performed to elucidate the in vivo role of IRF5 in tumorigenesis. Given its essential role in DNA damage-induced apoptosis, we explored the tumor suppressor function of IRF5 in IR-induced thymic lymphomagenesis. Somewhat surprisingly, we found that thymic lymphoma development was significantly suppressed in Irf5(-/-) mice as compared with wild-type littermates. Suppression was due, in part, to reduced thymocyte and HSC apoptosis, resulting in reduced compensatory proliferation, and reduced replication stress-associated DNA damage. The observed effects were independent of p53 or Puma as these proteins were upregulated in Irf5(-/-) mice in response to IR. This study demonstrates an important new role for IRF5 in maintaining HSC homeostasis after IR and supports the non-redundant functions of IRF5, p53 and PUMA in DNA damage-induced lymphomagenesis. We propose that IRF5 may be an attractive target for developing therapeutic agents to ameliorate radiation-induced bone marrow injury.

Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis

Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a “dicentric” chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2(YVMA) and ERBB2(G776VC), have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2(YVMA) to wild type using physiologically relevant peptide substrates reveals that ERBB2(YVMA) kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2(YVMA) phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis.

Cancer‐associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo

Global histone modification patterns correlate with tumor phenotypes and prognostic factors in multiple tumor types. Recent studies suggest that aberrant histone modifications play an important role in cancer. However, the effects of global epigenetic rearrangements on cell functions remain poorly understood. In this study, we show that the histone H3 lysine 9 (H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro. Overexpression of wild-type SUV39H1, but not enzymatically inactive SUV39H1, activated migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 trimethylation (H3K9me3) was specifically increased in invasive regions of colorectal cancer tissues. Moreover, the presence of H3K9me3 positively correlated with lymph node metastasis in colorectal cancer patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in a considerable decrease in survival rate. These data indicate that H3K9 trimethylation plays an important role in human colorectal cancer progression, possibly by promoting collective cell invasion.

Abstract 517: Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells.

Abstract The obscurins (∼70-820 kDa) are a family of proteins expressed from the 150 kb OBSCN gene, located on human chromosome 1. Sequencing analysis of breast and colorectal cancers, as well as melanoma and glioblastoma, revealed that obscurins are mutated at a substantial frequency and these mutations may be driving tumor formation. Our work has recently demonstrated that obscurins are present in normal breast, skin, and colon cells, but absent in their cancerous counterparts. Downregulation of obscurins’ expression is sufficient to allow non-tumorigenic breast epithelial cells to evade apoptosis induced by etoposide. Furthermore, absence of obscurins allows increased migration and invasion of breast epithelial cells. Remarkably though, we observed that ectopic expression of a fragment of obscurin composed of tandem src homology 3-Rho guanine exchange factor-pleckstrin homology (SH3-RhoGEF-PH) motifs was sufficient to decrease monolayer migration and invasion of metastatic breast cancer MDA-MB-231 cells, which are naturally deficient in obscurins, by >20% and >70%, respectively. Based on our observations that 1) absence of obscurins allows increased migration and invasion of breast cells, and 2) expression of an obscurin cassette mitigates this migratory and invasive behavior of metastatic breast cancer cells, we postulate that obscurins may act as metastasis suppressors in breast epithelium. We have therefore begun to investigate how the tripartite obscurin fragment, SH3-RhoGEF-PH, can influence the behavior of the cytoskeleton and therefore affect migration and invasion. We have found that the RhoGEF domain directly induces guanine nucleotide exchange in RhoA, but not Rac1 or Cdc42. This activity may be allosterically regulated by the adjacent SH3 domain, which interacts with the RhoGEF domain in a yeast two-hybrid assay. Furthermore, the PH domain binds to 3’-phosphorylated inositol lipid headgroups, providing a mechanism for the obscurins to respond to signals generated at the plasma membrane. Together, this evidence suggests that the tripartite signaling cassette can recapitulate the full-length obscurins’ metastasis-suppressing activity, and may merit development as a therapy for advanced breast cancer. Citation Format: Nicole A. Perry, Aikaterini Kontrogianni-Konstantopoulos. Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells. [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 517. doi:10.1158/1538-7445.AM2013-517

Abstract LB-42: In vivo screen for novel breast cancer promoting genes.

Abstract Mis-regulation of oncogenes and tumor suppressor genes causes cancer. Although the evolution of cancer is not conclusive, most evidence supports that tumors arise from collaboration of multiple genes. To better understand the process of breast tumor initiation and cancer progression we carried out an in vivo screen, for genes that promote tumor formation. Our screen was performed using lentiviral transduction of primary murine mammary epithelial cells, and orthotopic implantation into syngeneic recipient mice. Wild type mammary stem cells can regenerate an entire mammary ductal tree in the recipients. We chose a well-characterized ErbB2/Her2 over-expression mouse model as a source of donor cells because HER2 over-expression occurs in >20% of human breast cancer patients. A cDNA library consisting of a “druggable” set of ~1000 genes was screened in 12 subsets segregated by ORF length. Ten months after transplantation the recipient mice were euthanized and DNA from mammary gland tumors was harvested. No tumors formed in mice transplanted with cells into which a control virus had been transduced. Genes from the library that were expressed in the tumors were identified by sequencing. The ability of these genes to drive tumor formation was confirmed by transduction and transplantation into a second cohort of mice. One of the genes identified in this screen is GTF2I repeat domain containing 1 (GTF2IRD1). Multi-focal atypical hyperplasia formed in mammary glands over-expressing GTF2IRD1 while no hyperplasia was seen in the control mice. This effect depends on the expression of ErbB2 because expression of GTF2IRD1 itself in wild type mammary glands did not cause abnormal growth before the end point. Analysis of the cancer genome atlas database demonstrates that GTF2IRD1 expression is significantly increased in invasive breast cancer compared to normal controls. Therefore, our screen has identified a novel tumor-promoting gene that is of potential clinical importance. A major advantage of our method is the use of normal cells of origin and immunocompetent mice. Moreover, it could be expanded to genome-wide searches for new tumor- and metastasis-promoting genes. Citation Format: Yongliang Huo, Ian Macara. In vivo screen for novel breast cancer promoting genes. [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 LB-42. doi:10.1158/1538-7445.AM2013-LB-42

Metabolism in 'the driver's seat

It is increasingly accepted that metabolic changes in cancer cells can drive tumour formation. The finding that the SIRT6 protein suppresses tumour formation by regulating metabolism adds weight to this view.

Identification of Somatic Mutations in Parathyroid Tumors Using Whole-Exome Sequencing

The underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined. The aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing. Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas. Four of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing. This study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.

TSSC3 overexpression reduces stemness and induces apoptosis of osteosarcoma tumor-initiating cells

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents, typically presenting with poor prognosis. Recent studies suggested that tumor initiating cells (T-ICs) drive tumor formation and relapse or metastasis and are relatively resistant to cell death induced by conventional chemo- and radiotherapies. Therefore, the poor prognosis of OS appears to be associated with T-ICs. Here, we enriched T-ICs in OS cell lines and evaluated whether the imprinted gene TSSC3 (tumor-suppressing STF cDNA 3) associated with apoptosis could affect T-ICs in OS. Sarcosphere selection and serial clone-forming unit assays were successfully used to enrich T-ICs from OS cell lines. Enrichment of T-ICs from a malignantly transformed hFOB1.19 osteoblast cell line (MThFOB1.19) indicated that OS T-ICs could originate from differentiated cells, and most of these MThFOB1.19 cells showed stem-like features. TSSC3 was expressed at a low level in T-ICs, while overexpression of TSSC3 could efficiently downregulate the expression of stem cell markers Nanog, Oct4 and Sox2 in T-ICs and decrease the clone formation rate, as well as downregulate tumorigenesis in MThFOB1.19 cells, supporting a suppressive role for TSSC3 in OS T-ICs. Furthermore, overexpression of TSSC3 was found to induce apoptosis of OS T-ICs through increasing cleaved caspase-3 (active form), increasing the release of Cyt c and decreasing pro-caspase-9 (pro-enzyme form), as well as disruption of the mitochondrial membrane potential (ΔΨ). Taken together, our findings provide preliminary evidence that TSSC3 inhibits OS tumorigenicity through reducing stemness and promoting apoptosis of T-ICs. Thus, targeting TSSC3 may be a promising approach to suppressing tumorigenicity in OS.

Abstract 3491: Development of a new approach to kill non-small cell lung cancer cells with resistance to standard chemotherapy using parthenolide analogues

Abstract Lung cancer is the leading cause of cancer death in men and women worldwide. The poor prognosis of advanced non-small cell lung cancer (NSCLC) is due, in part, to emergence of tumor resistance to chemotherapy. Recent data indicate that human tumors including NSCLC contain a small subset of cancer stem/ progenitor cells (CSC) responsible for drug resistance and tumor maintenance. If such minute subsets of CSC drive tumor formation and drug resistance, therapies targeting the bulk tumor mass but not CSC will fail. We now confirm identification of subpopulations of chemotherapy-resistant human NSCLC cells with enrichment for CSC biomarkers and exhibiting significant CSC activity. We identified CD133+/ALDH+ tumor stem/progenitor cells from human lung cancer cells in vitro using established Aldefluor assays in combination with labeled anti-CD133 antibodies. Estrogen, a known risk factor for lung cancer progression, stimulated a modest increase in the numbers of CSC. In contrast to control CD133-/ALDH- tumor cell subsets, CSC subpopulations grew as tumor spheres and maintained self-renewal capacity in vitro and exhibited a greater tumorigenic capability than non-CSC subsets in vivo, properties indicative of CSC. Furthermore, resistance of CSC-like cells to cisplatin (a standard chemotherapy for NSCLC treatment) was fully reversed by treatment with parthenolide (PTL), a naturally-occurring sesquiterpene lactone compound with strong antitumor activity in leukemia and prostate cancer while sparing normal cells. The antitumor effect of PTL appears due to its action as a potent inhibitor of nuclear factor-βB (NF-κB) which is markedly activated by chemotherapy. To target CSC and suppress tumor progression, we synthesized and tested novel analogs of PTL with improved antitumor properties and aqueous solubility. PTL analogs inhibit proliferation of H157 NSCLC cells using both bulk cell preparations and CSC-subpopulations, with effects significantly different from control at P<0.05. Dose-dependent increments of PTL analogs increase apoptosis of CSC when compared with bulk cells. Moreover, PTL analogs inhibit cell proliferation of H23, A549 and H1975 NSCLC cells with known resistance to cisplatin (P<0.01). These compounds were able to sensitize cells to cisplatin-induced cytotoxicity (P<0.01) when cells were exposed to sub-optimal concentrations of cisplatin. Using Western blots, we find that PTL congeners inhibit phosphorylation of the p65 subunit of phospho-NF-κB and activation of IKKα/β. Thus, targeted inhibition of NF-κB may reverse tumor drug resistance by interfering with known NF-κB actions to regulate genes involved in proliferation, DNA damage response, antiapoptosis and angiogenesis. Further development of PTL analogs as therapeutics may lead to new strategies to treat NSCLC in the clinic. [Funded by CDMRP Lung Cancer Research Program LC 090297]. 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 3491. doi:1538-7445.AM2012-3491

Abstract 5073: Integrated genomics of high-resolution esophageal adenocarcinomas identify candidate driver genes in regions of large copy number aberrations

Abstract Introduction: Esophageal cancer occurs with two distinct histologies_ squamous cell carcinoma (SCC) and esophageal adenocarcinoma (EA). The incidence of EA has risen over the past few decades and is now the most common esophageal malignancy in the United States and Western Europe. The genome of esophageal adenocarcinoma is highly unstable due to the large number of amplification and deletion events. Presumably, such genomic events drive tumor formation and disease progression by altering the expression of genes within these regions. Several studies have reported large regions of genomic instability especially spanning chromosomal arms or in gene dense regions. Using a large patient cohort and high-resolution data for DNA copy number and expression, this study aims to identify potential driver genes and prognostic markers especially in whole chromosomal aberrations and gene dense regions. Materials & Methods: DNA copy number aberrations were explored in 116 esophageal adenocarcinomas (EA) using Affymetrix SNP 6.0 microarrays. Additionally, gene expression data from a subset of 110 of these tumors using Affymetrix U133 plus 2.0 microarrays were analyzed. DNA copy number aberrations were analyzed using Nexus 5.0 and GISTIC software while the gene expression data was processed in Partek Genomic Suite. Copy number and expression correlations for each gene in the regions of copy number aberrations were calculated using t-test to compare samples with the changes versus samples without the genomic aberrations. Results: We identified at least 50 regions of gains and losses of which 12 regions are either whole chromosomal arm aberrations or aberrations in gene dense regions. Some regions with large number genes include gains at 1q (460 genes), 7q21-22 (80 genes), 10q21-22 (100 genes), and losses at 4q (98 genes), 5q (283 genes), 21q (82 genes). Copy number and expression correlations for each gene in these regions considerably reduced the number of genes that could be considered as candidate driver genes. Furthermore, scouring the literature helped identify potential candidate driver genes such as ANP32E (1q), VCL (10q), CDKN2AIP (4q), and ING2 (4q). Additionally, we report identifying copy number and expression correlations of potential prognostic markers such as MCM4 (8q), CA9 (9p), KLF5 (13q) that lie in focal/smaller regions of genomic instability but at high frequencies. Conclusion: Analysis of the DNA copy number changes identified several regions of frequent gains and losses. At least 12 regions span the chromosomal arm or are in the gene dense regions. Using an integrated genomics approach with high-resolution DNA copy number and expression data, we are able to identify potential driver genes and prognostic markers that lie in these large amplification/deletion regions in EA. 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 5073. doi:1538-7445.AM2012-5073