Genes involved in senescence and immortalization.

Senescence is now understood to be the final phenotypic state adopted by a cell in response to several distinct cell physiological processes, including proliferation, oncogene activation and oxygen free radical toxicity. The role of telomere maintenance in immortalization and the roles of p16(INK4A), p19(ARF), p53 and other genes in senescence are being further elucidated. Significant progress continues to be made in our understanding of cellular senescence and immortalization.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

Separable portions of the CD2 cytoplasmic domain involved in signaling and ligand avidity regulation.

Effective T cell immune responses require the molecular interplay between adhesive and signaling events mediated by the T cell receptor for antigen (TCR) and other cell surface coreceptor molecules. In this report, we have distinguished between the role of regulated adhesion and transmembrane signaling in coreceptor function using the T cell glycoprotein CD2. By binding its ligands on antigen-presenting cell (APC), CD2 serves both to initiate signal transduction events and to promote cellular adhesion. Furthermore, the avidity of CD2 for one ligand, CD58 (LFA-3), is regulated by TCR signaling. We have expressed wild type CD2 and a series of mutated CD2 molecules in an antigen-specific murine T cell hybridoma. Structure-function studies using these stably transfected cell lines identify two structurally and functionally distinct regions of the 116 amino acid (aa) cytoplasmic domain. One region is required for CD2-mediated signal transduction, and a separate COOH-terminal 21 aa portion is required for CD2 activity regulation. Cell lines expressing CD2 molecules lacking the cytoplasmic segment required for CD2-initiated IL-2 production retain the ability to upregulate CD2 avidity. Conversely, cell lines expressing CD2 mutants lacking the cytoplasmic segment required for avidity regulation retain the ability to initiate CD2-specific signaling. In antigen-specific T cell responses, basal binding of CD2 to its ligands enhances antigen responsiveness only minimally, whereas regulated avidity and transmembrane signaling are both required for optimal coreceptor function. Taken together, these studies demonstrate the independent contributions of regulated adhesion and intracellular signaling in CD2 coreceptor function.

Mesosecrin: a secreted glycoprotein produced in abundance by human mesothelial, endothelial, and kidney epithelial cells in culture.

Human mesothelial cells, endothelial cells, and type II kidney epithelial cells growing in culture devote approximately 3% of their total protein synthesis to the production of an Mr approximately 46-kD, pI 7.1, secreted glycoprotein (designated Sp46). Fibroblasts make about 1/10th as much Sp46 as these cell types, and their synthesis is dependent upon hydrocortisone. Keratinocytes, urothelial cells, conjunctival epithelial cells, and mammary epithelial cells do not make detectable amounts of Sp46. Mesothelial cells secrete Sp46 onto the substratum, and from there it is subsequently released into the medium. Immunofluorescence analysis using specific antisera discloses that Sp46 is deposited beneath cells as a fine coating on the substratum. In sparse cultures, Sp46 is detected in trails behind motile cells. In contrast, secreted fibronectin coalesces into fibers, most of which remain in contact with and on top of the cells; thus Sp46 does not preferentially bind to fibronectin. About 6 kD of the mass of human Sp46 is N-linked oligosaccharide, which is terminally sialated before secretion. Sp46 has a low glycine content, indicating that it is not a collagenlike protein. Its NH2-terminal sequence over the first 40 amino acids does not resemble any protein for which sequence information is available. Sp46 appears to be a novel extracellular glycoprotein, high-level constitutive expression of which is restricted to mesoderm-derived epithelial and endothelial cells. We therefore propose for it the name "mesosecrin."

Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligand CD59.

The interaction of the T cell glycoprotein CD2 with one ligand, CD58, contributes to T cell function. We have identified CD59, a glycoprotein with complement-inhibitory function, as a second physiological ligand for CD2. Antibodies to CD59 inhibit CD2-dependent T cell activation in murine T cell hybridomas expressing human CD2. In an in vitro binding assay with purified CD58 and CD59, CD2+ cells bind not only immobilized CD58 but also CD59. With two complementary approaches, it was demonstrated that the binding sites on CD2 for CD58 and CD59 are overlapping but nonidentical. These observations suggest that direct interactions between CD2 and both CD58 and CD59 contribute to T cell activation and adhesion.

Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism.

The immortalization of human cells is a critical step in multistep carcinogenesis. Oral-esophageal carcinomas, a model system to investigate molecular mechanisms underlying squamous carcinogenesis, frequently involve cyclin D1 overexpression and inactivation of the p53 tumor suppressor. Therefore, our goal was to establish the functional role of cyclin D1 overexpression and p53 inactivation in the immortalization of primary human oral squamous epithelial cells (keratinocytes) as an important step toward malignant transformation. Cyclin D1 overexpression alone was found to induce extension of the replicative life span of normal oral keratinocytes, whereas the combination of cyclin D1 overexpression and p53 inactivation led to their immortalization. This study also demonstrates that immortalization of oral keratinocytes can be independent of telomerase activation, involving an alternative pathway of telomere maintenance (ALT).

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics.

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.

Mitogen stimulation cooperates with telomere shortening to activate DNA damage responses and senescence signaling.

Replicative senescence is induced by critical telomere shortening and limits the proliferation of primary cells to a finite number of divisions. To characterize the activity status of the replicative senescence program in the context of cell cycle activity, we analyzed the senescence phenotypes and signaling pathways in quiescent and growth-stimulated primary human fibroblasts in vitro and liver cells in vivo. This study shows that replicative senescence signaling operates at a low level in cells with shortened telomeres but becomes fully activated when cells are stimulated to enter the cell cycle. This study also shows that the dysfunctional telomeres and nontelomeric DNA lesions in senescent cells do not elicit a DNA damage signal unless the cells are induced to enter the cell cycle by mitogen stimulation. The amplification of senescence signaling and DNA damage responses by mitogen stimulation in cells with shortened telomeres is mediated in part through the MEK/mitogen-activated protein kinase pathway. These findings have implications for the further understanding of replicative senescence and analysis of its role in vivo.

Telomere and telomerase dynamics in human cells.

Accumulating evidence now implicates telomeres and telomerase as critical regulators genomic stability and replicative lifespan in mammalian cells. Disruption of telomere maintenance and/or telomerase expression contributes to the etiology of some degenerative diseases and may participate in the process of aging. Although telomere dysfunction and aberrant telomerase expression clearly play important roles in cancer development, the contribution of telomere biology to cancer is complex and involves both positive and negative influences on tumor development. Indeed, recent work from several laboratories suggests additional roles for telomeres and telomerase in both normal and malignant physiology. Understanding the complexity of telomere biology will provide further insights into chromosome biology in both normal and malignant cells.

Derivation of human tumor cells in vitro without widespread genomic instability.

The majority of adult human epithelial cancers exhibit evidence of genetic instability, and it is widely believed that the genetic instability manifested by aneuploidy or microsatellite instability plays an essential role in the genesis of these tumors. Indeed, most experimental models of cancer also show evidence of genomic instability. The resulting genetic chaos, which has widespread effects on many genes throughout the genome, confounds attempts to determine the precise cohort of genetic changes that are required for the transformation of normal human cells to a tumorigenic state. Here we show that genetic transformation of human kidney epithelial cells can occur in the absence of extensive aneuploidy, chromosomal translocations, and microsatellite instability. These observations demonstrate that the in vitro oncogenic transformation of human cells can proceed without widespread genomic instability.

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.

Characterization of HLA-A3-restricted cytotoxic T lymphocytes reactive against the widely expressed tumor antigen telomerase.

PURPOSE: We have reported previously that the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), is a widely expressed tumor-associated antigen recognized by CTLs. A nine-amino acid peptide derived from hTERT binds strongly to HLA-A2 antigen and elicits CTL responses against a broad panel of hTERT+ tumors (but not hTERT+ hematopoietic progenitor cells). The applicability of hTERT as a potential target for anticancer immunotherapy would be widened by the identification of epitopes restricted to other common HLA alleles, such as HLA-A3 antigen. EXPERIMENTAL DESIGN: Using a method of epitope deduction, HLA-A3-restricted peptide epitopes were screened from hTERT and tested for immunogenicity in a human in vitro T-cell system. RESULTS: The hTERT peptide K973 was used to generate specific CD8+ CTLs from HLA-A3+ cancer patients and healthy individuals. These CTLs lysed hTERT+ tumors from multiple histologies in an MHC-restricted fashion, suggesting that the epitope is naturally processed and presented by tumors. In contrast, highly enriched HLA-A3+ CD34+ peripheral blood progenitor cells or activated T cells were not lysed. CONCLUSION: Given the expression of HLA-A2 and HLA-A3 antigen in the general population, these findings extend the potential applicability of hTERT as a therapeutic target to >60% of all cancer patients. The characterization of hTERT as a polyepitope, polyallelic tumor-associated antigen may provide an approach for circumventing therapy-induced resistance potentially mediated by antigenic- and allelic-loss tumor escape mutants.

TERT promoter mutations and monoallelic activation of TERT in cancer.

Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer.

TRAF2 is an NF-κB-activating oncogene in epithelial cancers.

Aberrant nuclear factor (NF)-κB activation is frequently observed in human cancers. Genome characterization efforts have identified genetic alterations in multiple components of the NF-κB pathway, some of which have been shown to be essential for cancer initiation and tumor maintenance. Here, using patient tumors and cancer cell lines, we identify the NF-κB regulator, TRAF2 (tumor necrosis factor (TNF) receptor-associated factor 2), as an oncogene that is recurrently amplified and rearranged in 15% of human epithelial cancers. Suppression of TRAF2 in cancer cells harboring TRAF2 copy number gain inhibits proliferation, NF-κB activation, anchorage-independent growth and tumorigenesis. Cancer cells that are dependent on TRAF2 also require NF-κB for survival. The phosphorylation of TRAF2 at serine 11 is essential for the survival of cancer cells harboring TRAF2 amplification. Together, these observations identify TRAF2 as a frequently amplified oncogene.

PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

The complete sequences of plasmids pFNeo and pMH-Neo: convenient expression vectors for high-level expression of eukaryotic genes in hematopoietic cell lines.

The eukaryotic expression vector, pFNeo, confers resistance to the antibiotic G418 and directs hematopoietic-specific expression of proteins under the control of the long terminal repeat from the Friend spleen focus-forming virus. Here, we report the entire nucleotide sequence of pFNeo. We have also constructed and sequenced a pFNeo-based expression vector (pMH-Neo) that carries an improved multiple cloning site region for easier subcloning. The utility of both vectors was demonstrated by transfection of murine T-cell hybridomas by electroporation. Transfection with either pFNeo or pMH-Neo yielded a high frequency (1 in 2 x 10(4)) of G418-resistant cell lines.

SV40 early region oncoproteins and human cell transformation.

We now understand neoplastic transformation to be the consequence of multiple acquired genetic alterations. The combination of these acquired changes confer the various phenotypes that constitute the clinical features of cancer. Although only rare human cancers derive from a viral etiology, the study of DNA tumor viruses that transform rodent and human cells has led to a greater understanding of the molecular events that program the malignant state. In particular, investigation of the viral oncoproteins specified by the Simian Virus 40 Early Region (SV40 ER) has revealed critical host cell pathways, whose perturbation play an essential role in the experimental transformation of mammalian cells. Recent work has re-investigated the roles of two SV40 ER oncoproteins, the large T antigen (LT) and the small t antigen (ST), in human cell transformation. Co-expression of these two oncoproteins, together with the telomerase catalytic subunit, hTERT, and an oncogenic version of the H-Ras oncoprotein, suffices to transform human cells. LT inactivates two key tumor suppressor pathways by binding to the retinoblastoma protein (pRB) and p53. The ability of ST to transform human cells requires interactions with PP2A, an abundant family of serine-threonine phosphatases. Here we review recent developments in our understanding of how these two viral oncoproteins facilitate human cell transformation.

An integrative analysis reveals functional targets of GATA6 transcriptional regulation in gastric cancer.

Lineage-restricted transcription factors (TFs) are frequently mutated or overexpressed in cancer and contribute toward malignant behaviors; however, the molecular bases of their oncogenic properties are largely unknown. As TF activities are difficult to inhibit directly with small molecules, the genes and pathways they regulate might represent more tractable targets for drug therapy. We studied GATA6, a TF gene that is frequently amplified or overexpressed in gastric, esophageal and pancreatic adenocarcinomas. GATA6-overexpressing gastric cancer cell lines cluster in gene expression space, separate from non-overexpressing lines. This expression clustering signifies a shared pathogenic group of genes that GATA6 may regulate through direct cis-element binding. We used chromatin immunoprecipitation and sequencing (ChIP-seq) to identify GATA6-bound genes and considered TF occupancy in relation to genes that respond to GATA6 depletion in cell lines and track with GATA6 mRNA (synexpression groups) in primary gastric cancers. Among other cellular functions, GATA6-occupied genes control apoptosis and govern the M-phase of the cell cycle. Depletion of GATA6 reduced the levels of the latter transcripts and arrested cells in G2 and M phases of the cell cycle. Synexpression in human tumor samples identified likely direct transcriptional targets substantially better than consideration only of transcripts that respond to GATA6 loss in cultured cells. Candidate target genes responded to the loss of GATA6 or its homolog GATA4 and even more to the depletion of both proteins. Many GATA6-dependent genes lacked nearby binding sites but several strongly dependent, synexpressed and GATA6-bound genes encode TFs such as MYC, HES1, RARB and CDX2. Thus, many downstream effects occur indirectly through other TFs and GATA6 activity in gastric cancer is partially redundant with GATA4. This integrative analysis of locus occupancy, gene dependency and synexpression provides a functional signature of GATA6-overexpressing gastric cancers, revealing both limits and new therapeutic directions for a challenging and frequently fatal disease.

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling.

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK MAPK pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified p21-activated kinase 1 (PAK1) as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 30--33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation.

Emerging roles for the non-canonical IKKs in cancer.

The IκB Kinase (IKK)-related kinases TBK1 and IKKɛ have essential roles as regulators of innate immunity by modulating interferon and NF-κB signaling. Recent work has also implicated these non-canonical IKKs in malignant transformation. IKKɛ is amplified in ∼30% of breast cancers and transforms cells through the activation of NF-κB. TBK1 participates in RalB-mediated inflammatory responses and cell survival, and is essential for the survival of non-small cell lung cancers driven by oncogenic KRAS. The delineation of target substrates and downstream activities for TBK1 and IKKɛ has begun to define their role(s) in promoting tumorigenesis. In this review, we will highlight the mechanisms by which IKKɛ and TBK1 orchestrate pathways involved in inflammation and cancer.