Aleksandra Dakic

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.

HPV E6 protein interacts physically and functionally with the cellular telomerase complex

Telomerase activation is critical for the immortalization of primary human keratinocytes by the high-risk HPV E6 and E7 oncoproteins, and this activation is mediated in part by E6-induction of the hTERT promoter. E6 induces the hTERT promoter via interactions with the cellular ubiquitin ligase, E6AP, and with the c-Myc and NFX-1 proteins, which are resident on the promoter. In the current study we demonstrate that E6 protein interacts directly with the hTERT protein. Correlating with its ability to bind hTERT, E6 also associates with telomeric DNA and with endogenous active telomerase complexes. Most importantly, E6 increases the telomerase activity of human foreskin fibroblasts transduced with the hTERT gene, and this activity is independent of hTERT mRNA expression. Unlike its ability to degrade p53, E6 does not degrade hTERT protein in vitro or in vivo. Our studies of E6/hTERT interactions also reveal that the C-terminal tagged hTERT protein, although incapable of immortalizing fibroblasts, does immortalize keratinocytes in collaboration with the viral E7 protein. Thus, E6 protein mediates telomerase activation by a posttranscriptional mechanism and these findings provide a model for exploring the direct modulation of cell telomerase/telomere function by an oncogenic virus and suggest its potential role in both neoplasia and virus replication.

HPV E7 contributes to the telomerase activity of immortalized and tumorigenic cells and augments E6-induced hTERT promoter function

The E6 and E7 proteins of high-risk HPVs are both required for the immortalization of primary human keratinocytes and the maintenance of the malignant phenotype of HPV-positive cancer cell lines. Our previous studies have shown that E6 protein binds Myc protein and that both E6 and Myc associate with and cooperatively activate the hTERT promoter, thereby increasing cellular telomerase activity. In this study, we evaluated the role of E7 in the maintenance and activation of telomerase in immortalized and tumorigenic cells. siRNA knockdown of either E6 or E7 (or both) in HPV-immortalized cells or an HPV-positive cancer cell line reduced hTERT transcription and telomerase activity. Since telomerase was inhibited by E7 siRNA in cells that independently expressed the E6 and E7 genes, our results reveal an independent role for E7 in the maintenance of telomerase activity. However, E7 alone was insufficient to increase endogenous hTERT mRNA or telomerase activity, although it significantly augmented E6-induced hTERT transcription and telomerase activity. To further explore this apparent E7-induced promoter augmentation, we analyzed an exogenous hTERT core promoter in transduced keratinocytes. E7 alone induced the wt hTERT promoter and augmented E6-induced hTERT promoter activity. Mutation of the E2F site in the hTERT promoter abrogated the ability of E7 to induce the hTERT promoter or to enhance the ability of E6 to induce the promoter. Correspondingly, keratinocytes expressing E6 and a mutant E7 (defective for binding pRb pocket proteins) showed lower telomerase activity than cells expressing wt E6 and wt E7. Thus, HPV E7 plays a role in the maintenance of telomerase activity in stable cell lines and augments acute, E6-induced hTERT promoter activity.

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.

Cell-Restricted Immortalization by Human Papillomavirus Correlates with Telomerase Activation and Engagement of the hTERT Promoter by Myc

ABSTRACT The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.

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.

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

Significance Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from noncancer cells remains an ongoing challenge, and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anticancer targets. We determined that the key oncoprotein of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing that are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.

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.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patients prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.