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Supplementary Movie from Tumorigenic Conversion of Primary Human Esophageal Epithelial Cells Using Oncogene Combinations in the Absence of Exogenous Ras

Supplementary Figure and Movie Legends from Tumorigenic Conversion of Primary Human Esophageal Epithelial Cells Using Oncogene Combinations in the Absence of Exogenous Ras

Supplemental Material & Methods

To investigate pathways of human esophageal squamous cell transformation, we generated esophageal tumor cells using human telomerase– and SV40-immortalized primary esophageal epithelial cells (EPC2) by overexpression of selected combinations of oncogenes. H-Ras, c-Myc, or Akt, but not epidermal growth factor receptor (EGFR), induced transformed colonies in soft agar. By contrast, bioluminescence imaging of genetically altered immortalized esophageal cells revealed that Akt, EGFR, or H-Ras, but not c-Myc, resulted in tumor formation in immunodeficient mice. H-Ras-driven tumors showed highly tumorigenic phenotypes with 2.6 ± 0.6 days for doubling, whereas Akt and EGFR tumors doubled every 9.5 ± 1.6 and 6.1 ± 1.2 days, respectively. H-Ras-driven tumors expressed the hypoxia-inducible factor target Glut1, whereas Akt- or EGFR-driven tumors had evidence of angiogenesis and no detectable Glut1 expression. Proliferation rates among these tumors were similar, but there was reduced apoptosis in the more aggressive H-Ras-driven tumors that also developed aneuploidy and multiple centrosomes. c-Myc overexpression did not result in tumorigenic conversion but introduction of Bcl-XL into c-Myc-expressing cells generated tumors. Although cytokeratin expression was typical of squamous carcinoma, gene expression profiling was done to compare the four different types of engineered tumors with human esophageal squamous cell carcinomas and adenocarcinomas. Interestingly, c-Myc plus Bcl-XL transformants mimicked squamous carcinomas, whereas H-Ras-, EGFR-, and Akt-driven tumors were similar to adenocarcinomas in their molecular profiles. These genetically engineered models may provide new platforms for understanding human esophagus cancer and may assist in the evaluation of new therapies. (Cancer Res 2006; 66(21): 10415-24)

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5+ crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies. Cancer Res; 76(3); 700–12. ©2015 AACR.

Supplementary Figures 1-2, Tables 1-2 from Tumorigenic Conversion of Primary Human Esophageal Epithelial Cells Using Oncogene Combinations in the Absence of Exogenous Ras

Supplementary Figure Legends for Supplementary Figures 1-9.

Supplementary Figures 1-9. Sup. Fig. 1: IV injections of the monoclonal DR5-targeting MD5-1 antibody in combination with fluorouracil (5-FU) and CPT-11 sensitizes mice to chemotherapy-induced toxicity. Area-under-the curve (AUC) analysis of weight loss curves following different doses of fluorouracil (5-FU). Sup. Fig. 2: High dose treatment with the DR5-targeting antibody MD5-1 in combination with low-dose CPT-11 triggers liver toxicity whereas low dose MD5-1-treatment combined with high dose chemo triggers GI-toxicity in mouse strains known to be resistant to TRAIL-R-targeting. Sup. Fig. 3: Targeting of DR5 in combination with radiochemotherapy does not trigger synergistic bone marrow toxicity. Sup. Fig. 4: DR5-targeting sensitizes human small intestinal epithelial cells to cell death following chemotherapy. Sup. Fig. 5: DR5-targeting in combination with chemotherapy triggers cell death in the crypts of the GI tract in mice in vivo. Sup. Fig. 6: P53-dependent gene expression changes in the mouse colonic epithelium following fluorouracil and irinotecan. Affymetrix gene expression mRNA data on p53-responsive genes in the colon of mice treated with 5-FU (A) and CPT-11 (B). Sup. Fig. 7: Chemotherapy-dependent reliance on cell cycle regulating- and apoptosis-signaling genes for the toxic response following chemotherapy and DR5-targeting. Sup. Fig. 8: Chk2 is a modulator GIT following combined chemotherapy and DR5-targeting. Sup. Fig. 9: Pharmacologic targeting of Chk2 countermeasure GIT without impacting the anti-tumor response following chemotherapy and DR5-targeting.

The five-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained a dismal 9% for approximately 40 years with an urgent need for novel therapeutic interventions. ONC201 is the founding member of the imipridone class, comprised of orally bioavailable small molecules that have shown efficacy in multiple tumor types both in animal models and in Phase I/II clinical trials. ONC201 is a potent inducer of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. TRAIL is an innate immune mechanism which induces programmed cell death of cancer cells. We observed that PDAC cells upregulated ATF4, CHOP, and DR5 after treatment with ONC201. This occurred in cell lines that are susceptible to ONC201-induced apoptosis and in ones that are not. In response to ONC201, PDAC cells downregulated anti-apoptotic proteins including c-FLIP, BclXL, XIAP, cIAP1, and survivin. We hypothesized that TRAIL receptor agonists might induce selective, synergistic apoptosis in pancreatic cancer cell lines treated with ONC201. We screened 7 pancreatic cancer cell lines and found synergy with ONC201 and rhTRAIL or the novel TRAIL receptor agonist TLY012 in 6 of the 7 cell lines tested. In vivo experiments using BxPC3 and HPAFII xenograft models showed that the combination of ONC201 plus TLY012 significantly delays tumor growth as compared to controls. Immunohistochemical analysis of the tumors after three doses of the combination showed significantly increased cleavage of caspase 3 in vivo as compared to controls. Taken together, the preclinical efficacy of ONC201 and TLY012 represents a novel therapeutic option for further testing in pancreatic cancer patients. This combination showed marked efficacy in tumor cells that are both sensitive and resistant to the pro-apoptotic effects of ONC201, providing rationale to further investigate the combination of ONC201 plus TLY012 in patients with pancreatic cancer.

Greater than 10 million patients succumb to cancer each year. Cancer metastasis is responsible for more than 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. As a seed for metastases, circulating tumor cells (CTCs) present a new dimension and horizon for clinical doctors in diagnosis, prognosis prediction, treatment monitoring, disease mechanism, and drug development. CTCs could gradually replace tissue biopsies which are painful and may be difficult to obtain depending on tumor location. CTC isolation is feasible after minimally invasive liquid biopsy and provides the basis for a multitude of ex vivo and in vivo studies including establishment of CTCs-derived 2D and 3D cultures. Organoids are miniscule models of tissues that grow in a 3D semisolid extracellular matrix medium with specific growth factors supplied. CTCs-derived 3D organoids play a vital role in precision oncology because they can preserve tumor heterogeneity, imitate the tumor microenvironment (TME), mimic cancer hypoxia in the TME, and maintain cancer and metastasis phenotypes. Cytotoxic CD8+ T cells are the most powerful effectors in the anticancer immune response. We hypothesized that co-culture of CTCs-derived 3D organoids and autologous cytotoxic CD8+ T cells could maximize patient-relevance of laboratory assessment of cancer-treatment immune-system interactions to facilitate precision oncology practice. TellBio’s novel TellDx CTC technology allows for isolation of viable and intact CTCs in liquid biopsies, regardless of cancer type. Unlabeled CTCs were cultured in growth factor reduced Matrigel with organoid culture WENRAS medium. Magnetic beads labeled white blood cells (WBCs) were cultured with T cell culture medium - WBCs and magnetic beads usually separate in three days - cytotoxic CD8+ T cells were isolated with the EasySep™ Human CD8+ T Cell Isolation Kit. CTCs-derived 3D organoids and autologous cytotoxic CD8+ T cells were co-cultured with or without different drug treatments - cytotoxicity was measured with CellTiterGlo® 3D Cell Viability Assay and imaging, and further mechanistic studies were feasible. Our co-culture platform enables us to utilize a patient's peripheral blood or pleural effusions to create a patient-specific, in vivo-like TME and immune microenvironment to model and assess ex vivo responses to investigational and FDA-cleared cancer therapies, and potentially provide oncologists with insights to improve clinical outcomes. Citation Format: Lanlan Zhou, Leiqing Zhang, Lindsey Carlsen, Kelsey E. Huntington, Vida Tajiknia, Andrew George, Arielle De La Cruz, Arunasalam Navaraj, Praveen Srinivasan, Maximilian Schwermann, Benedito A. Carneiro, Wafik S. El-Deiry. Co-culture of circulating tumor cells (CTCs)-derived 3D organoids and autologous cytotoxic CD8+ T cells: A new functional precision oncology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6706.

Our Laboratory was established in 1994 at Univ. of Pennsylvania. Lab members demonstrated initial competencies by performing cell culture, western blots, immunofluorescence, and flow cytometry showing induction of p53/p21(WAF1) in cells treated with chemotherapy. Years later, our Laboratory of Translational Oncology & Experimental Cancer Therapeutics moved to Penn State Univ., Fox Chase Cancer Center/Temple Univ. and then Brown Univ. By 2020, with desire for inclusiveness (everyone succeeds), scientific rigor/reproducibility mandated by NIH, and as a training and mentoring activity (lab scientists/trainees/students mentoring others at High School level and beyond), we established a process for onboarding and training new cancer researchers. By Fall of 2022, there were 17 current Brown University undergraduate students (10 receiving research credit and 7 not receiving credit), HS students, 7 graduate students (PhD, masters, MD/PhD), and 6 medical students working with collaborating faculty at our laboratory at Brown’s Legorreta Cancer Center. After completion of biosafety training, and required trainings such as by IACUC, new lab members complete basic competencies in cell culture, cell viability, and western blot analysis that include technical, presentation quality output, and quantitative/statistical rigor to satisfy current standards for journal publication. For cell culture this includes pathogen free conditions, authentication, attention to details of routine procedures, documentation of morphology, freezing, thawing, passaging, seeding density, and managing cell populations to not run out of cells. Cell viability assessment includes attention to culture conditions, synergy analysis, data robustness, and presentation, and for western blots attention to quality of blots, protein quantification, loading, labeling, antibody specificity and sensitivity controls, presentation at 2022 standards, conventions for splicing, and issues with reproducibility including biological replicates, and generalizability. Additional and advanced competencies include RT-PCR, long-term colony assays, 3-D cultures (spheroids, organoids), transfection (overexpression, knockdown, CRISPR), co-culture and triculture with immune cells and fibroblasts, cytokine profiling, in vivo studies, in vivo imaging, immunohistochemistry, flow cytometric analysis, single cell techniques, viral infection, circulating tumor cell isolation, blood immune and cytokine analysis, and work with transgenic organoids and inducible cancer predisposing alleles. Modeling the tumor microenvironment, relevance to human cancer and translational directions are emphasized. Shared online lab resources, protocols, practices, videos, and manuscripts are available for lab members. The framework herein may be of interest to others involved in similar training programs. Citation Format: Wafik S. El-Deiry, Andrew George, Francesca Di Cristofano, Praveen Srinivasan, Lindsey Carlsen, Kelsey E. Huntington, Arielle De La Cruz, Leiqing Zhang, Marina Hahn, Shuai Zhao, Attila Seyhan, Bradley D. DeNardo, Aaron W. Maxwell, Dae Hee Kim, Alex Raufi, Hina Khan, Stephanie L. Graff, Don S. Dizon, Christopher Azzoli, Abbas E. Abbas, Roxanne Wood, Rishi R. Lulla, Howard P. Safran, Benedito A. Carneiro, Arunasalam Navaraj, Xiaobing Tian, Shengliang Zhang, Lanlan Zhou. Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4185.

Hepatocellular carcinoma (HCC) and colorectal cancer liver metastases (CRCLM) are leading causes of death worldwide, with therapeutic options limited to surgical resection and trans-arterial chemoembolization (TACE). TACE is a minimally-invasive approach to induce cancer cell death through a local hypoxia-dependent mechanism, and intrahepatic catheterization further enables the delivery of therapeutic agents to the tumor microenvironment (TME). Phase 1 clinical trials are ongoing for treatment of primary liver cancer and uveal melanoma with liver metastases using pressure-enabled drug delivery of SD-101 in combination with systemic immune checkpoint inhibitors (ICIs). Since acidosis is associated with immunosuppression in the TME, pH modulating drugs (PMDs) may restore physiologic conditions and augment treatment response for patients undergoing TACE with immunotherapy. Pre-clinical studies using PMDs with ICIs or adoptive immune cell transfer have demonstrated increased tumor lymphocyte infiltration, increased survival, and reduced tumor volume compared to ICI treatment or T cell infusion alone. High-throughput fluorescent imaging of HCC or CRC lines co-cultured with TALL-104 cells indicates increased cancer cell death with PMD treatment alone, while imaging studies of combination PMD and ICI treatment are underway. In addition, Luminex 200 cytokine profiling of Hep3B indicates that the NHE1 inhibitor cariporide decreases HCC production of IL-8 and GDF-15 under normoxia and the proton pump inhibitor lansoprazole increases GDF-15 and MIF and decreases B2M, IL-8, CXCL10, and CXCL5. Combination PMD and ICI studies are needed to inform drug combinations and dosages in syngeneic murine models of primary liver cancer and CRCLM. Subcutaneous and orthotopic tumor models are expected to demonstrate the efficacy of local PMD treatment with systemic immunotherapy. Citation Format: Ryan Malpass, Kelsey E. Huntington, Thomas Brown, Lanlan Zhou, Leiqing Zhang, Arunasalam Navaraj, Wafik S. El-Deiry, Aaron Maxwell. Combination treatment with pH modulating drugs and immune checkpoint inhibitors increases TALL-104 cytotoxicity in preclinical co-culture experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2373.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19- (+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M- CSF, IL-1a, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.

Oncogenic mouse double minute 2 homolog (MDM2) is an E3-ubiquitin ligase that facilitates proteasomal degradation of p53. MDM2 amplification occurs in cancer and has been implicated in accelerated tumor growth, known as hyper-progression, following immune-checkpoint therapy. MDM2 amplification also predicts poor response to immune-checkpoint inhibitors. We sought to evaluate the role of MDM2 in T-cell-mediated immune resistance. Ovarian clear cell carcinoma cell lines carrying wild-type p53 with low/high MDM2 expression were investigated in a T-cell co-culture system evaluating T-cell-mediated tumor killing. Targeting of MDM2 was achieved by siRNA transfection or a selective MDM2 inhibitor, AMG-232 and tumor cells were tested in the T-cell co-culture system. AMG-232 activated p53 signaling in cancer cells and relative resistance to AMG-232 was observed in high MDM2-expressing cell lines. Cell lines with high MDM2 expression were more resistant to T cell-mediated tumor killing. Targeting MDM2 by gene-silencing or pharmacological blockade with AMG-232 enhanced T-cell killing of cancer cells. AMG-232 potentiated tumor cell killing by T-cells in combination with anti-PD-1 antibody treatment, regardless of changes in PD-L1 expression. The AMG-232 was not toxic to the T-cells. MDM2 inhibition lowered expression of Interleukin-6, a pro-inflammatory pro-tumorigenic cytokine. Our data support targeting MDM2 in tumors with overexpression or amplification of MDM2 as a precision therapy approach to overcome drug resistance including hyper-progression in the context of immune checkpoint therapy.

Heterogeneity in the p53 response to traditional chemotherapy is recognized but its significance has yet to be exploited in the clinic. Further investigation could provide insight into the relative importance of individual p53 target genes and identify unique characteristics of specific drugs, guiding therapy selection. Wild-type or p53 null HCT-116 cells were treated with oxaliplatin, cisplatin, CPT-11 (irinotecan), or 5-Fluorouracil (5-FU) at an IC50 dose for 8 hours, in triplicate. Total cellular RNA was isolated and Clariom™ D microarrays were used to measure changes in RNA expression relative to untreated cells. TAC software was used to calculate fold-changes and statistical significance. Western blot was used to confirm treatment-induced increase in p53 and to further investigate p53 target heterogeneity at the protein level. The top 100 p53-dependent genes upregulated (FC>1.5) by each drug were identified, which revealed BTG2 as the top upregulated gene after oxaliplatin, cisplatin, and 5-FU treatment. Eight genes appeared in all four lists (GPR87, FAS, TP53INP1, DCP1B, POLH, PDGFA, MDM2, and PHLDA3) but variations in magnitude of change across drugs were noted for several including GPR87, FAS, TP53INP1, and MDM2. Moreover, heterogeneity in the magnitude of change of p53 targets was observed by western blot, which also showed differential upregulation of p21 and DR5 across drug treatment conditions. The lists contained numerous genes that were unique to each treatment, with oxaliplatin regulating the highest number of unique genes (31/100). Several transcripts outside of the top 100 were also identified as unique in each drug treatment including BAX, which showed slight preferential upregulation by 5-FU. Evaluation of the p53-independent transcriptional regulation revealed a strikingly significant and unique regulation of histone modifications by 5-FU compared to the other treatments. Beyond the specific genes mentioned, variation in the magnitude of change and identification of the unique targets after each drug treatment highlights potentially important mechanistic differences between the chemotherapeutic drugs. The appearance of eight transcripts in the top 100 upregulated genes for all four drug treatments indicates their importance as well as the contribution of the p53 response to the molecular effects of chemotherapy. We are currently further evaluating the relevance of these genes and their unique contributions to the action of the chemotherapeutic drugs. Citation Format: Lindsey Carlsen, Lanlan Zhou, Liz J. Hernández Borrero, Arunasalam Navaraj, Shengliang Zhang, Christoph Schorl, Wafik S. El-Deiry. The transcriptional response in colorectal cancer cells varies across four clinically used chemotherapeutic drugs in terms of gene identity, magnitude of change, and p53 dependence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 999.

// Prashanth Gokare 1, 2 , Arunasalam Navaraj 2 , Shengliang Zhang 1, 2 , Noboru Motoyama 3 , Shen-Shu Sung 4 , Niklas K. Finnberg 1, 2 1 Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Medical Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA 2 Penn State Hershey Cancer Institute, Penn State Hershey Medical Center, Hershey, PA 17104, USA 3 Institute of Longevity, Department of Cognitive Brain Sciences Research Institute, National Center for Geriatrics and Gerontology, Aichi 474-8511, Japan 4 Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17104, USA Correspondence to: Niklas K. Finnberg, e-mail: niklas_finnberg@hotmail.com Keywords: Chk2, topoisomerase inhibitors, apoptosis, etoposide, myelosuppression Received: September 29, 2015 Accepted: March 28, 2016 Published: April 18, 2016 ABSTRACT The DNA damage response (DDR) gene cell cycle checkpoint kinase 2 (Chk2) triggers programmed cell death and lethal radiation-induced toxicity in mice in vivo . However, it is not well established to what extent targeting of Chk2 may protect from dose-limiting toxicities (DLT) inflicted by mainstay cancer chemotherapy. We screened different classes of chemotherapy in wild type and Chk 2 -deficient cells. Here we show that loss of Chk2 protect from cell death in vitro and lethal toxicity in vivo following treatment with topoisomerase II (TOP2)–inhibitors whereas no such protection was observed following treatment with topoisomerase I (TOP1) inhibitors. Furthermore, through combined in silico and functional screens of the Diversity Set II (NCI/NTP) chemical library we identified the carbanilide-derivative NSC105171, also known as ptu-23, as a novel Chk2 inhibitor (Chk2i). Indeed, NSC105171 can be administered safely to mice to countermeasure etoposide-induced toxicity. Incorporation of Chk2i into chemotherapy protocols employing TOP2-inhibitors may be an effective strategy to prevent DLT’s without interfering with treatment.

The original HTML version of this article was updated shortly after publication following a formatting issue which resulted in the abstract heading being omitted. This error has now been corrected in the HTML version of the Article. The PDF version of the Article was correct at the time of publication.

Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10-20 mM for 1-2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3-4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo.

To investigate the role of tumor suppressors BRca1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53 gene-specific knockdowns. Stable knockdown of BRCA1 alone in MCF10A cells led to centrosome amplification, impaired p53 protein stability, increased sensitivity towards DNa-damaging agents, defective chromosomal condensation at mitosis and elevated protein levels of cyclin D1 and c-myc. While over-expression of mutant H-Ras transformed MCF10A cells, depletion of BRCA1 dramatically enhanced the in vivo tumorigenesis that was associated with higher levels of VEGF, enhanced vascularization and less apoptosis in the BRCA1-deficient Ras-transformed tumors. The Ras-transformed BRCA1-deficient tumors exhibited features of the epithelial-to-mesenchymal transition, appeared to secrete matrix metalloproteases as visualized by in vivo bio-imaging of tumors using fluorescent probe MMP680, and were locally metastatic to lymph nodes. Our results suggest that loss of BRca1 function may contribute to the aggressiveness of Ras-MAPK driven human breast cancer with associated increase in levels of cyclin D1 and c-myc, enhanced MAPK activity, angiogenic potential & invasiveness. This mammary xenograft tumor model may be useful as a tool to understand human breast tumor angiogenesis and metastasis, as well as to test candidate therapeutics.

Some naturally occurring strains of fungi cease growing through successive subculturing, i.e., they senesce. In Neurospora, senescing strains usually contain intramitochondrial linear or circular plasmids. An entire plasmid or its part(s) integrates into the mtDNA, causing insertional mutagenesis. The functionally defective mitochondria replicate faster than the wild-type mitochondria and spread through interconnected hyphal cells. Senescence could also be due to spontaneous lethal nuclear gene mutations arising in the multinucleated mycelium. However, their phenotypic effects remain masked until the nuclei segregate into a homokaryotic spore, and the spore germinates to form a mycelium that is incapable of extended culturing. Ultimately the growth of a fungal colony ceases due to dysfunctional oxidative phosphorylation. Results with senescing nuclear mutants or growth-impaired cytoplasmic mutants suggest that mtDNA is inherently unstable, requiring protection by as yet unidentified nuclear-gene-encoded factors for normal functioning. Interestingly, these results are in accord with the endosymbiotic theory of origin of eukaryotic cells.

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5+ crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies. Cancer Res; 76(3); 700–12. ©2015 AACR.

We recently showed that p300/CREB-binding protein (CBP) plays an important role in maintaining cells in G0/G1 phase by keeping c-myc in a repressed state. Consistent with this, adenovirus E1A oncoprotein induces c-myc in a p300-dependent manner, and the c-myc induction is linked to S-phase induction. The induction of S phase by E1A is dependent on its binding to and inactivating several host proteins including p300/CBP. To determine whether there is a correlation between the host proteins binding to the N-terminal region of E1A, activation of c-myc and induction of S phase, we assayed the c-myc and S-phase induction in quiescent human cells by infecting them with Ad N-terminal E1A mutants with mutations that specifically affect binding to different chromatin-associated proteins including pRb, p300, p400 and p300/CBP-associated factor (PCAF). We show that the mutants that failed to bind to p300 or pRb were severely defective for c-myc and S-phase induction. The induction of c-myc and S phase was only moderately affected when E1A failed to bind to p400. Furthermore, analysis of the E1A mutants that fail to bind to p300, and both p300 and PCAF suggests that PCAF may also play a role in c-myc repression, and that the two chromatin-associated proteins may repress c-myc independently. In summary, these results suggest that c-myc deregulation by E1A through its interaction with these chromatin-associated proteins is an important step in the E1A-mediated cell cycle deregulation and possibly in cell transformation.

Although treatments have improved patient prognosis in surgically resectable colorectal cancer, new effective drugs with improved safety profiles are needed to improve the currently poor outcomes of patients with recurrent or metastatic colorectal cancer. Quinacrine, a small molecule anti-malarial agent that has activity in giardiasis, lupus, prion disease, and used as a means of non-surgical sterilization, has shown cytotoxic activity across a broad range of cancers. Here, we evaluate the potential of adding quinacrine to anticancer chemotherapeutics and targeted agents as a potential novel combinatorial therapy for advanced colon cancer. We show that quinacrine synergizes with 5-fluorouracil and significantly enhances the cytotoxicity of sorafenib in a panel of 10 human colorectal cancer cell lines, including those with KRAS mutations protein gel blot analysis confirmed that quinacrine's anticancer activity partially arises from its ability to stabilize p53 and lower anti-apoptotic protein levels. In a series of in vivo studies, quinacrine monotherapy lowered the tumor load of nu/nu mice bearing human colorectal cancer xenografts. In combination, quinacrine and 5-Fluorouracil significantly delayed tumor growth of a variety of different xenografts, as compared to each agent administered alone. Our results suggest that the administration of quinacrine in combination with chemotherapeutic agents and targeted agents should be further explored in patients with recurrent, locally advanced, or metastatic colorectal cancer.

Some naturally occurring strains of fungi cease growing through successive subculturing, i.e., they senesce. In Neurospora, senescing strains usually contain intramitochondrial linear or circular plasmids. An entire plasmid or its part(s) integrates into the mtDNA, causing insertional mutagenesis. The functionally defective mitochondria replicate faster than the wild-type mitochondria and spread through interconnected hyphal cells. Senescence could also be due to spontaneous lethal nuclear gene mutations arising in the multinucleated mycelium. However, their phenotypic effects remain masked until the nuclei segregate into a homokaryotic spore, and the spore germinates to form a mycelium that is incapable of extended culturing. Ultimately the growth of a fungal colony ceases due to dysfunctional oxidative phosphorylation. Results with senescing nuclear mutants or growth-impaired cytoplasmic mutants suggest that mtDNA is inherently unstable, requiring protection by as yet unidentified nuclear-gene-encoded factors for normal functioning. Interestingly, these results are in accord with the endosymbiotic theory of origin of eukaryotic cells.

We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. These results suggested that a defective S-phase checkpoint may contribute to enhanced sensitivity of Plk2-deficient cells to replication stress. Consistent with this hypothesis, we observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted. We also observed higher levels of Chk1 protein in Plk2-deficient cells that were associated with reduced levels of Serine 317-phosphorylated Chk1. In aphidicolin-treated cells, there were lower levels of Serine 317-phosphorylated Chk1 when Plk2 was knocked-down. Plk2 was demonstrated to interact with Chk2, Chk1, Serine 317-phosphorylated Chk1 and p53. Thus, increased cell death observed after aphidicolin treatment and release in Plk2-deficient cells may result from both higher levels of replication stress-induced DNA damage and a dysfunctional S-phase checkpoint.

Current ongoing clinical trials are exploring combined targeting of the TRAIL-death receptor DR5 with radiochemotherapy. It is not clear mechanistically why targeting of the DR5 increasingly sensitizes cancer cells to cell death without affecting cells in normal tissues. Preclinical data suggests that integration of DR5 agonist antibodies into chemotherapy protocols has the promise to significantly improve cancer specific responses without negatively impacting dose-limiting toxicities. Here we show that targeting of DR5 in mice with the mouse-specific mAb MD5-1 in combination with chemotherapy triggers synergistic gastrointestinal toxicities (GIT) that is associated with the death of Lgr5+ crypt basal columnar (CBC) cells. This type of GIT is mediated by p53 and in the case of irinotecan, the cell cycle checkpoint kinase 2 (Chk2). Pharmacologic targeting of Chk2 sensitized syngeneic mouse colorectal cancer tumors lacking P53 to the irinotecan/MD5-1 combination while at the same time protecting the gastrointestinal tract from injury. Our data indicates that blockade of cell cycle checkpoint kinases can help prevent GIT triggered by a combination of DNA damaging chemotherapy and DR5 agonists. Pharmacologic targeting of Chk2 can improve the anti-tumor response of such drug combinations and subsequently provides a rationale on how to improve therapeutic indices of DR5-targeting in the context of chemotherapy usage. Citation Format: Niklas K. Finnberg, Prashanth Gokare, Arunasalam Navaraj, Krystle A. Lang Kuhs, George Cerniglia, Hideo Yagita, Kazuyoshi Takeda, Noboru Motoyama, Wafik S. El-Deiry. DR5-targeting sensitizes Lgr5+ stem cells to p53 and Chk2-dependent chemotherapy-induced cell death and produces dose-limiting gastrointestinal toxicity (GIT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2015-2926

To investigate pathways of human esophageal squamous cell transformation, we generated esophageal tumor cells using human telomerase– and SV40-immortalized primary esophageal epithelial cells (EPC2) by overexpression of selected combinations of oncogenes. H-Ras, c-Myc, or Akt, but not epidermal growth factor receptor (EGFR), induced transformed colonies in soft agar. By contrast, bioluminescence imaging of genetically altered immortalized esophageal cells revealed that Akt, EGFR, or H-Ras, but not c-Myc, resulted in tumor formation in immunodeficient mice. H-Ras-driven tumors showed highly tumorigenic phenotypes with 2.6 ± 0.6 days for doubling, whereas Akt and EGFR tumors doubled every 9.5 ± 1.6 and 6.1 ± 1.2 days, respectively. H-Ras-driven tumors expressed the hypoxia-inducible factor target Glut1, whereas Akt- or EGFR-driven tumors had evidence of angiogenesis and no detectable Glut1 expression. Proliferation rates among these tumors were similar, but there was reduced apoptosis in the more aggressive H-Ras-driven tumors that also developed aneuploidy and multiple centrosomes. c-Myc overexpression did not result in tumorigenic conversion but introduction of Bcl-XL into c-Myc-expressing cells generated tumors. Although cytokeratin expression was typical of squamous carcinoma, gene expression profiling was done to compare the four different types of engineered tumors with human esophageal squamous cell carcinomas and adenocarcinomas. Interestingly, c-Myc plus Bcl-XL transformants mimicked squamous carcinomas, whereas H-Ras-, EGFR-, and Akt-driven tumors were similar to adenocarcinomas in their molecular profiles. These genetically engineered models may provide new platforms for understanding human esophagus cancer and may assist in the evaluation of new therapies. (Cancer Res 2006; 66(21): 10415-24)

DNA repair defects can predispose to cancer development and progression. We previously showed that the breast and ovarian cancer susceptibility gene product BRCA1, through p53, upregulates expression of the XPE gene DDB2 encoding the nucleotide excision repair protein p 48. Both XPE and XPC are p53 target genes containing p53 response elements. To further explore the role of BRCA1 and p53 in repair of photoproducts, we eliminated wild type p53 from U2OS osteosarcoma cells and found that cyclobutane pyrimidine dimer (CPD) repair was markedly impaired following UV damage whereas repair of 6-4 photoproduct (6-4 PP) occurred efficiently. Overexpression of p53 in p53-null Calu-6 cells also enhanced CPD repair. In HCC1937 breast cancer cells, harboring mutant BRCA1 and p53 genes, repair of CPD was markedly impaired. Reintroduction of either p53 or BRCA1 using adenovirus vectors into HCC1937 alone had little effect on repair of CPD whereas the combination of p53 and BRCA1 resulted in efficient repair of CPD. Thus there appears to be a cooperative effect between p53 and BRCA1 that may involve induction of repair proteins, inhibition of p53-induced cell death by BRCA1 with altered p53 selectivity towards repair pathways and/or p53-independent effects of BRCA1 on CPD repair.

p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two highly homologous, conserved transcriptional coactivators, and histone acetyltransferases (HATs) that link chromatin remodeling with transcription. Cell transformation by viral oncogene products such as adenovirus E1A and SV40 large T antigen depends on their ability to inactivate p300 and CBP. To investigate the role of p300 in cell-cycle progression, we constructed stable rat cell lines, which conditionally overexpress p300 from a tetracycline-responsive promoter. When p300 was induced in these cells, serum-stimulated S-phase entry was significantly inhibited. The inhibition of S-phase induction was associated with down-regulation of c-Myc , but not of c-Fos or c-Jun . Simultaneous overexpression of c-Myc and p300 before serum stimulation reversed the inhibition of S-phase induction to a significant level, indicating that the inhibition of c-Myc to a large extent is responsible for the p300 inhibition of G 1 exit. Similar studies with stable rat cell lines that overexpress a mutant p300, which lacks the HAT activity, showed that the intrinsic HAT activity of p300 is not required for the negative regulation of c-Myc or G 1 . These findings, and our previously published results (Kolli, S., Buchmann, A. M., Williams, J., Weitzman, S. & Thimmapaya, B. (2001) Proc. Natl. Acad. Sci. USA 98, 4646–4651), establish an important negative regulatory role for p300 in c-Myc expression that may be important in maintaining the cells in the G 0 /G 1 phase of the cell cycle.

TO THEEDITOR: We read with interest the article by Iinuma et al 1 that describes their study examining the clinical significance of cancer stem cells in the peripheral blood of patients with colorectal cancer (CRC). The defining characteristics of a circulating cancer stem cell (CTSC) are its capacity for self-renewal and for initiation of distant metastases; some of these cells are also resistant to traditional chemo- therapy. 2,3 The concept that circulating tumor stem cells can be iden- tified and targeted is attractive and has major diagnostic, prognostic, and therapeutic implications for patients with metastatic cancer. However, the identification of authentic CTSCs continues to be a challenge because of the lack of a clear understanding of the biologic heterogeneity of tumor stem-cell populations and also because of the lack of characterization of surface markers that predict clinically im- portant subsets of CTSCs, such as those with aggressive malignant potential or drug resistance.

Navaraj, A., Pandit, A., and Maheshwari, R. 2000. senescent: A new Neurospora crassa nuclear gene mutant derived from nature exhibits mitochondrial abnormalities and a “death” phenotype. Fungi are capable of potentially unlimited growth. We resolved nuclear types from multinuclear mycelium of a phenotypically normal wild isolate of the fungus Neurospora intermedia by plating its uninucleate microconidia and obtained a strain which, unlike the “parent” strain, exhibited clonal senescence in subcultures. The mutant gene, senescent, was introgressed into N. crassa and mapped four map units to the right of the his-1 locus on linkage group VR. senescent is the first nuclear gene mutant of Neurospora derived from nature that shows the death phenotype. Death of the sen mutant occurred faster at $34^o C$ than at 22 or $26^o C$. Measurements of oxygen uptake of conidia using respiratory inhibitors and the spectrophotometric analyses of mitochondrial cytochromes showed that in sen cultures grown at $34^o C$, cytochromes b and $aa_3$ were present but cytochrome c was absent. By contrast at $26^o C$, cytochromes b and c were present but cytochrome $aa_3$ was diminished in the late subcultures. This suggested that the sen mutation does not affect the potential to produce functional cytochromes. The deficiency of the respiratory chain cytochromes may not be the cause of death of the sen mutant because the cytochrome c and $aa_3$ mutants of N. crassa are capable of sustained growth whereas sen is not. Possible explanations for the observations are discussed.

Hepatocellular carcinoma (HCC) accounts for approximately 600,000 deaths per year worldwide. Effective therapeutic strategies for advanced stage patients are lacking, and the median survival rate for patients who are not eligible for surgical resection is a mere 6 months. Circulating tumor cells (CTCs) are detectable in the peripheral blood of cancer patients, are accessible through a minimally invasive procedure, and may provide a source of tumor tissue for analyzing molecular markers of therapeutic response that may help guide treatment protocols. To demonstrate this potential, we developed a system for imaging and quantifying multiplexed molecular markers of response to sorafenib/mapatumumab combination therapy in an HCC CTC model. The sorafenib/mapatumumab combination regimen is currently in phase 2 clinical trials for the treatment of HCC, and molecular markers that mediate the synergistic anti-tumor activity of this combination have been identified. These markers include phospho-p42/44 MAPK (p-ERK), Mcl-1, c-IAP2, and apoptotic markers such as cleaved caspase-3. We developed a technical approach using MultiSpectral Imaging (MSI) to quantitatively measure multiplexed fluorescent signals from primary antibodies specific to phospho-ERK, Mcl-1, c-IAP-2, and cleaved caspase-3 that had been conjugated to quantum dots (Qdots) with distinct fluorescence emission spectra. We first confirmed the ability to modulate marker expression and uncouple multiplexed Qdot signals by MSI in cultured HCC cells that had been treated with sorafenib or sorafenib/mapatumumab combinations. We then applied this detection system to HCC cells that were spiked into human blood and recovered using FDA-approved CTC isolation technology (CellSearch CTC Test by Veridex). Our data demonstrate the potential for studying molecular markers of therapeutic response in CTCs. Information gained from this technology may be used to guide therapeutic decisions or serve as pharmacokinetic/pharmacodynamic endpoints in experimental clinical protocols. 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 5665. doi:1538-7445.AM2012-5665

Breast cancer is the most frequent malignancy among women, causing at least 373,000 deaths per year worldwide, and in United States alone an estimated 208,000 women were diagnosed with breast cancer in 2010. Recent studies indicate that breast cancer is initiated by breast cancer stem cells (BCSCs) that express CD44+/high CD24-/low surface markers. Mammospheres are non-adherent spherical cell clusters grown in selective culture conditions. Our experimental results using mammosphere cultures of different breast cancer cells such as MDA-MB-231 show drastic enrichment of breast cancer stem-like cells with the phenotype of CD44+/high CD24-/low by flow cytometry analysis. We have implanted mammosphere-derived cells into mammary fat pads of nude mice and sampled blood from such tumor-bearing animals to look for the presence of unique breast cancer cells with metastatic potential {circulating tumor cells (CTCs)}, by scoring for Epcam+/cytokeratin+/CD45- tumor cells using the FDA-approved Veridex CellSearch Sysytem. As these metastatic breast cancer cells stably express ZS-Green protein, isolated ZS-Green positive CTCs are being further subjected to breast cancer-specific multiplexed molecular marker analysis. When a mouse with the metastatic breast cancer cell line - 4T1, stained with the CellVue dye is implanted in mammary fat pads, high levels of metastasis are detected with the CellVue marker in different internal organs such as the lungs, liver, heart and lymph nodes. Fluorescence microscopy analysis of blood withdrawn from animals bearing orthotopic 4T1 breast xenograft tumors, could detect ZS-Green positive mouse breast cancer CTCs. These mouse animal models bearing xenograft tumors are facilitating our understanding of CTC detection as well as analysis methods and also in understanding of human breast cancer metastasis. Open questions we are addressing include heterogeneity of CTCs, relationships in stem cell markers and prognostic markers when CTCs are compared with the primary tumors, relationships between tumor burden and tumor aggressiveness with CTC numbers in this mouse model. In addition the model provides an opportunity for testing candidate therapeutic agents and drug combinations. 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 1400. doi:1538-7445.AM2012-1400

Colorectal cancer (CRC) is the third leading cause of death due to cancer in USA. Restoration of wild-type (wt) p53 function alone has been shown to cause tumor regression in mice with minimal toxicity. p53-restoration compounds may provide a tumor-specific therapeutic regimen to overcome the resistance and toxicity associated with current CRC therapy. Utilizing a p53-responsive luciferase reporter-based screen we identified the fluorescent red bacterial pigment Prodigiosin as a potent p53 family transcriptional activator. Anti-tumoral properties of Prodigiosin have been reported for various types of cancer both in vitro and in vivo with minimal toxicity to normal tissues but the p53 mechanism has not been previously recognized. Prodigiosin has been shown to induce mitochondria-dependent apoptosis in cancer cells, but the role of the pro-apoptotic protein Bax in anti-tumor efficacy of Prodigiosin in CRC has not been clearly defined. Using fluorescence microscopy we determined that Prodigiosin localizes to the mitochondria in CRC cells. Results of trypan-blue dye exclusion tests reveal that HCT116 wild-type and Bax-null cells were equally sensitive to Prodigiosin-induced cell death. Thus, Bax played a minor role in Prodigiosin-induced cell death. The p53 family transcriptional activation by Prodigiosin is independent of the p53 status of the CRC cells. In vivo Prodigiosin induced p53 transcriptional activity and reduced tumor volume in subcutaneous xenografts of CRC cells. Prodigiosin induced apoptosis, inhibited cell proliferation and suppressed angiogenesis in vivo in CRC cells. Emerging evidence suggests that CRC tumors contain a rare population of CRC stem cells (CSCs) capable of self-renewal that are responsible for long-term sustenance of the tumor, local tumor recurrence and metastatic relapse. CSCs are therapy resistant since they are dormant and possess various efflux mechanisms. We hypothesize that anti-tumor effects of Prodigiosin involve targeting of the CSC population by p53 restoration in CSCs. With flow cytometric analysis for Prodigiosin fluorescence we determined that Prodigiosin is not effluxed from CRC cells. We have previously demonstrated that efflux of calcein AM is a marker for the cancer stem cell population. Here, we report that Prodigiosin is retained by the calcein low population (CloP) and induced apoptosis in CloP, as determined by fluorescence microscopy. Prodigiosin suppressed the ability of CRC cells to be serially passaged in vitro. Ongoing work involves determination of: mechanism of anti-angiogenic effect of Prodigiosin; effect of Prodigiosin on CSC frequency, self renewal and tumorigenicity; synergy of Prodigiosin with chemotherapeutic drugs for CRC in targeting CSCs. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. 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 1170. doi:1538-7445.AM2012-1170

Breast cancer is the most frequent malignancy among women, causing 373,000 deaths per year worldwide. Recent studies indicate that breast cancer is initiated by breast cancer stem cells (BCSCs) which express CD44+/high CD24−/low surface markers. Mammospheres are non-adherent spherical cell clusters grown in selective culture conditions. Our experimental results using mammosphere cultures of different metastatic, human breast cancer cells such as MDA-MB-231 and MDA-MB-468, showed drastic enrichment of breast cancer stem-like cells with the phenotype of CD44+/high CD24−/low in flow cytometry analysis, with specific antibody conjugates. Using the FDA-approved Veridex CellSearch System, we successfully recovered Epcam+/cytokeratin+/CD45− breast cancer cells from 7.5 ml of human blood to which a known number of MDA-MB-468 or H-Ras transformed MCF10A cells had been added. When these recovered breast cancer cells were subjected to Q-Dot conjugated p-ERK antibody staining and quantification, signal intensity clearly demonstrated higher levels of nuclear p-ERK in MCF10A-Ras cells whereas immunostaining for surface EGFR expression was much stronger in MDA-MB-468 cells. We are modeling mammosphere-derived breast cancer cells enriched for stem cell markers by orthotopic implantation into mammary fat pads of nude mice, sampling blood from tumor-bearing animals to detect the presence of Circulating Tumor Cells (CTCs) using the Veridex CellSearch System and further for breast cancer specific multiplexed marker analysis. These stem cell marker enriched xenografted human breast tumors are facilitating our understanding of cellular signaling pathways active in CTCs, and also in understanding of the role of cancer stem cells in human breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-57. doi:10.1158/1538-7445.AM2011-LB-57