Vera Simic

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

ABSTRACT The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.

Radiation Induces Diffusible Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to Conditionally Reprogram and Immortalize Epithelial Cells

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

Quantitative Measurement of Human Papillomavirus Type 16 E5 Oncoprotein Levels in Epithelial Cell Lines by Mass Spectrometry

ABSTRACT The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.

ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes.

The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death.

Abstract 4256: Functional analysis for cancer precision medicine using patient-derived 2D and 3D cell models

Background: Preclinical models provide an essential tool to study both cancer biology with particular relevance to identifying appropriate therapies and new targets. Both Patient-derived xenografts and 3D methods require significant time for the necessary amplification of the tumor cells, which limits clinical utility. Even classical 2D cultures suffer from their long time requirements for establishing a cell line with a low efficiency (1-30%). Conventional cancer cell lines usually fail to reflect the complex genotypes and phenotypes of the corresponding primary tumor. Recently, we described the use feeder cells and a ROCK inhibitor to induce the conditional reprogramming (CR) of adult epithelial cells into a basal or stem-like state. The induction of these CR cells is reversible, and the removal of feeders and ROCK inhibitor, coupled with their placement in environments that mimick their native environment (Matrigel, air-liquid interface (ALI), and the renal capsule in mice) allows cells to differentiate normally. Importantly, the CR technology can generate 2×106 cells in a week from small biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. A recent study utilized the CR method to initiate cultures from CT-guided lung biopsies and identify combination of therapies (Science 2014). Three recent reviews in Nature series highlighted CR method as one of the next-generation patient-derived cancer models (Nat Rev Clin Oncol, 2014, Nat Rev Genetics and Nat Rev Cancer 2015). Primary goal: The clinical utility and standard protocols for generating patient CR cultures. We therefore initiated study to examine whether CR cultures reflected the biology and genotype of the original tumor and whether cultures might be used to predict clinical responses. Procedures and Results: We first worked out standard protocols for clinical sampling, storage, shipping, freezing and the preparation of conditioned medium. CR methods were then used to generate matched cultures from both tumor cells and adjacent normal cells from patients with prostate cancer or lung cancer. CR cultures were established efficiently from these tumors (>95% for prostate and lung cancer CRCs). Following the rapid establishment of CR cultures, they were then transferred 3D cultures. The data with Matrigel 3D and (ALI) demonstrated that CR cells reexpressed cell specific markers and were well-differentiated. We also characterized them for their growth properties, induction of tumors in immunodeficient mice, karyotype, and their exome and transcriptome profile. Conclusion: CR cells, coupled with sequential 3D culture conditions, appears to provide optimal conditions for inducing the differentiation of normal cells and for differentiating normal from tumor cells. Finally, this cell-based approach should be useful for defining the functional heterogeneity of the respective primary tumors and for evaluating appropriate therapies. Citation Format: Xuefeng Liu, Ewa Krawczyk, Ogla Timofeeva, Nancy Palechor-Ceron, Aleksandra Dakic, Vera Simic, Bhaskar Kallakury, Anatoly Dritschilo, Richard Schlegel. Functional analysis for cancer precision medicine using patient-derived 2D and 3D cell models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4256.

Abstract 5145: Y-27632 inhibits Myc-induced apoptosis and cooperates with Myc to immortalize human keratinocytes

The high-risk HPV E6 and E7 genes are sufficient to immortalize human foreskin keratinocytes (HFKs) through activation of telomerase and inactivation of the RB/p16 pathway, respectively. Our previous work has shown that Myc (via induction of hTERT) may substitute for E6 and immortalize HFKs when combined with HPV E7. When expressed alone, however, Myc induced p53 expression and apoptosis. In the current study we found that Myc, in combination with the ROCK inhibitor (Y-27632), led immortalization of human foreskin keratinocytes. Our data confirmed that Myc induced apoptosis and blebbing of human keratinocytes when cultured in serum free synthetic medium (KGM) and that Y-27632 rapidly inhibits this process. Thus, our data demonstrate that Myc is capable of independently immortalizing HFKs when apoptosis pathways are blocked. Citation Format: Aleksandra Dakic, Kyle Divito, Shuang Fang, Nancy Palechor-Ceron, Vera Simic, Sujata Choudhury, Songtao Yu, Cynthia M. Simbulan-Rosenthal, Dean Rosenthal, Richard Schlegel, Xuefeng Liu. Y-27632 inhibits Myc-induced apoptosis and cooperates with Myc to immortalize human keratinocytes. [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 5145. doi:10.1158/1538-7445.AM2015-5145

Abstract 2972: Conditionally reprogrammed cells (CRCs): A new model for cancer research and personalized medicine

Background: Current cancer cell lines often fail to reflect the genotypes and phenotypes of the tumors from which they were derived due to the accumulation of genetic and epigenetic alterations during passage in vitro. This obviously limits the ability to use such cell lines for predicting responses to drug-, radiation-, or immuno-therapies. Until recently, it remained a challenge to rapidly and efficiently generate cell cultures from individual patients in a time-limited manner that allowed for choosing appropriate therapies. Last year we described the use feeder cells and a ROCK inhibitor to induce the conditional reprogramming of adult epithelial cells into a basal or stem-like (AJP, 2012, 2013; NEJM, 2012, PNAS 2012). Cultures generated from normal tissue, referred to as conditionally reprogrammed cells (CRCs), do not express high levels of proteins characteristic of iPSCs or ESCs such as Sox2, Oct4, Nanog, or Klf4. More important, the induction of CRCs is reversible, and the removal of feeders and ROCK inhibitor allows cells to differentiate normally to tissue origin. The CRC technology can generate 2×10 6 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Primary goal: The clinical utility of CRCs for patient care needs to be established. We therefore initiated studies to examine whether CRCs established from tumors would reflect the biology and genotype of the original tumor and whether the tumor CRCs could be used to predict clinical responses. Procedures: We used CRC methods to generate matched cultures from both tumor cells and adjacent normal cells from cancer patients and relevant mouse models. We characterized these CRC lines for their growth properties, induction of tumors in immunodeficient mice, karyotype, differentiation, and transcriptome profile. We also compared the sensitivity of the matched CRCs to commonly used chemotherapy drugs. Results: The CRC technique efficiently established cell cultures from human and rodent tumors. For example, we established matched normal and tumor CRCs for a patient with a squamous carcinoma of the tongue. The tumor CRC from this patient exhibited a highly abnormal karyotype and harbored a mutation of the p53 gene. The matched normal CRC exhibited a normal karyotype and wild p53. The tumor CRCs, but not the normal CRCs, efficiently induced squamous cell carcinomas when injected subcutaneously into nude mice. Similarly, we generated 4 cultures from pancreatic cancers, 3 of which exhibited mutations in the Ras gene. We were also able to utilize the CRC method to define an effective therapy for patient with an aggressive lung papillomatosis. Finally, our studies indicate that we are able to generate micro-heterogenous tumor CRCs from a small biopsy. Conclusion: CRCs show promise for evaluating tumor responses to selected therapies and for defining the functional heterogeneity of their respective primary tumors. Citation Format: Xuefeng Liu, Ewa Krawczyk, Nancy Palechor-Ceron, Weisheng Wang, Hang Yuan, Aleksandra Dakic, Vera Simic, Bhaskar Kullakury, Priscilla Furth, Richard Schlegel. Conditionally reprogrammed cells (CRCs): A new model for cancer research and personalized medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2014-2972