Retroviral Oncogenes Research Articles

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.

A History of Cancer Research: Retroviral Oncogenes.

The discovery and characterization of retroviral oncogenes were important milestones in cancer research. The viruses turned out not to be key causes of cancer in humans, but the oncogenes they carried provided key clues to the role cellular genes, the proto-oncogene counterparts of these sequences, played in tumorigenesis. In this excerpt from his forthcoming book, Joe Lipsick looks back at early work on retroviruses, such as the experiments that distinguished their ability to infect and transform cells, the groundbreaking work on Src, and some of the controversy surrounding the Nobel Prize awarded for these discoveries.

NF-κB and Human Cancer: What Have We Learned over the Past 35 Years?

Transcription factor NF-κB has been extensively studied for its varied roles in cancer development since its initial characterization as a potent retroviral oncogene. It is now clear that NF-κB also plays a major role in a large variety of human cancers, including especially ones of immune cell origin. NF-κB is generally constitutively or aberrantly activated in human cancers where it is involved. These activations can occur due to mutations in the NF-κB transcription factors themselves, in upstream regulators of NF-κB, or in pathways that impact NF-κB. In addition, NF-κB can be activated by tumor-assisting processes such as inflammation, stromal effects, and genetic or epigenetic changes in chromatin. Aberrant NF-κB activity can affect many tumor-associated processes, including cell survival, cell cycle progression, inflammation, metastasis, angiogenesis, and regulatory T cell function. As such, inhibition of NF-κB has often been investigated as an anticancer strategy. Nevertheless, with a few exceptions, NF-κB inhibition has had limited success in human cancer treatment. This review covers general themes that have emerged regarding the biological roles and mechanisms by which NF-κB contributes to human cancers and new thoughts on how NF-κB may be targeted for cancer prognosis or therapy.

Stem Cell Theory of Cancer: Implications of a Viral Etiology in Certain Malignancies.

In 1911, Peyton Rous (Nobel Prize winner in 1966) demonstrated that a virus (i.e., RSV) caused cancer in chickens. In 1976, Bishop and Varmus (Nobel Prize winners in 1989) showed that the cellular origin of retroviral oncogenes was actually normal cellular genes (i.e., proto-oncogenes). In this article, we revisit the role viruses play in the genetic origin of cancer. We review a link between viruses or cancer and autoimmunity in an alternative stem cell origin of cancer. We propose that a virus is more likely to cause cancer when it infects a progenitor stem-like cell rather than a progeny differentiated cell. We postulate that both known (e.g., HBV and HPV) and novel viruses (e.g., SARS-CoV-2) pose an imminent threat in the emergence of chronic viral diseases as well as virally induced malignancies. Knowing the origin of cancer has profound implications on our current conception and perception of cancer. It affects our conduct in cancer research and our delivery of cancer care. It would be ironic if viruses turn out to be a useful tool and an ideal means in our quest to verify a genetic versus stem cell origin of cancer. When it pertains, oncology recapitulates ontogeny; although genetic makeup is pivotal, cellular context may be paramount to elucidating a stem cell origin of cancer.

Proteomic Analysis of Src Family Kinase Phosphorylation States in Cancer Cells Suggests Deregulation of the Unique Domain.

The Src family kinases (SFK) are homologs of retroviral oncogenes, earning them the label of proto-oncogenes. Their functions are influenced by positive and negative regulatory tyrosine phosphorylation events and inhibitory and activating intramolecular and extramolecular interactions. This regulation is disrupted in their viral oncogene counterparts. However, in contrast to most other proto-oncogenes, the genetic alteration of these genes does not seem to occur in human tumors and how and whether their functions are altered in human cancers remain to be determined. To look for proteomic-level alterations, we took a more granular look at the activation states of SFKs based on their two known regulatory tyrosine phosphorylations, but found no significant differences in their activity states when comparing immortalized epithelial cells with cancer cells. SFKs are known to have other less well-studied phosphorylations, particularly within their unstructured N-terminal unique domains (UD), although their role in cancers has not been explored. In comparing panels of epithelial cells with cancer cells, we found a decrease in S17 phosphorylation in the UD of Src in cancer cells. Dephosphorylated S17 favors the dimerization of Src that is mediated through the UD and suggests increased Src dimerization in cancers. These data highlight the important role of the UD of Src and suggest that a deeper understanding of proteomic-level alterations of the unstructured UD of SFKs may provide considerable insights into how SFKs are deregulated in cancers. IMPLICATIONS: This work highlights the role of the N-terminal UD of Src kinases in regulating their signaling functions and possibly in their deregulation in human cancers.

Integrated computational approaches on pyrazoline derivatives as B-Raf kinase inhibitors for the development of novel anticancer agents

Raf kinases (rapidly accelerated fibrosarcoma) is related to retroviral oncogenes found in three different isoforms, A, B and C-Raf and played a significant part in mediating cell survival and proliferation. Various derivatives of B-Raf inhibitors are developed under laboratory level and some are under clinical trial studies. Here, we completed different computational techniques to identify features required for B-Raf kinase inhibitory activity by using 49 pyrazoline analogues. The development of pharmacophore, 3D QSAR analysis, virtual screening and docking studies were used for the development of novel compounds. For 3-dimentional-QSAR study, all compounds were divided into training and test set and determined the importance of substituents on 49 pyrazoline derivatives for B-Raf kinase inhibitory activity. The final models showed significant statistical values (R2 = 0.8325, Q2 = 0.5854) for atom based QSAR and (R2 = 0.7945, Q2 = 0.5808) for field based QSAR by using 3 partial least squares (PLS) factor. The validation results of 3D QSAR models showed R2 CV = 0.625, 0.5803, for both models (atom based and field based), consecutively. The docking studies of potent pyrazoline analogues (C1, C12, C14, C16n) showed good XP docking scores (-7.962, -8.864, -6.893, -7.171) with the interaction of amino acid residue such as PHE 583, CYS 532, SER 536 and ASP 594. The compounds ZINC13989686 and ZINC23908357 generated through virtual screening study showed their best docking scores -11.97 and -10.73 with receptor B-Raf kinase (PDB ID:2FB8). These data may be used for the development of potent compounds against B-Raf kinase.

Enzootic nasal tumor virus type 2 envelope of goats acts as a retroviral oncogene in cell transformation.

Enzootic nasal tumor virus type 1 (ENTV-1) (ovine nasal tumor virus) and ENTV-2 (caprine nasal tumor virus) are known to be causative agents of enzootic nasal adenocarcinoma (ENA) in sheep and goats, respectively. Although the nucleotide and amino acid sequences of ENTV-1 and ENTV-2 are quite similar, they are recognized as phylogenetically distinct viruses. The envelope protein of ENTV-1 functions as an oncoprotein in the in vitro transformation of epithelial cells and fibroblasts. Thus, it is the primary determinant of in vivo tumorigenesis in ENA. As per our knowledge, no previous studies have reported in detail the role of ENTV-2 in ENA tumorigenesis. Here, in order to investigate the molecular mechanism of caprine ENA oncogenesis by ENTV-2, we have attempted to identify the transforming potential of ENTV-2 envelope, and investigated the activation of cell signaling pathways in oncogenic transformation. Our findings confirmed that ENTV-2 envelope was capable of inducing oncogenic transformation of rat cell lines in vitro. Further, we found that MAPK, Akt, and p38 were constitutively activated in ENTV-2 envelope-transformed clone cells. In addition, inhibitor experiments revealed that MEK-MAPK and PI3K-Akt signaling pathways are involved in the ENTV-2 envelope-induced cell transformation. These data indicate that ENTV-2 envelope could induce oncogenic transformation by signaling pathways that are also utilized by ENTV-1 envelope.

RAS mutations in human cancers: Roles in precision medicine

Ras proteins play a crucial role as a central component of the cellular networks controlling a variety of signaling pathways that regulate growth, proliferation, survival, differentiation, adhesion, cytoskeletal rearrangements and motility of a cell. Almost, 4 decades passed since Ras research was started and ras genes were originally discovered as retroviral oncogenes. Later on, mutations of the human RAS genes were linked to tumorigenesis. Genetic analyses found that RAS is one of the most deregulated oncogenes in human cancers. In this review, we summarize the pioneering works which allowed the discovery of RAS oncogenes, the finding of frequent mutations of RAS in various human cancers, the role of these mutations in tumorigenesis and mutation-activated signaling networks. We further describe the importance of RAS mutations in personalized or precision medicine particularly in molecular targeted therapy, as well as their use as diagnostic and prognostic markers as therapeutic determinants in human cancers.

Of oncogenes and open science: an interview with Harold Varmus.

Harold Varmus has made pioneering contributions to our understanding of cancer as a genetic disease. The discovery of the cellular origin of retroviral oncogenes earned him and his long-term collaborator, Michael Bishop, the Lasker Prize for Basic Medical Sciences in 1982 and the Nobel Prize in Physiology and Medicine in 1989. Throughout his career, Varmus has held several leadership roles that shaped science policy in the US and worldwide, and he has been an outspoken advocate for open science. In this interview, he talks (among other things) about the factors that shaped his early career choices, the thrill of scientific discovery, and the importance of including diverse populations in genomic studies of cancer and other diseases.

PIK3CA in cancer: The past 30 years.

Almost thirty years ago, PI3K was discovered as a lipid kinase associated with certain oncoproteins. The first decade of research on PI3K saw the identification, purification and cloning of PI3Kα. The second decade of research was noted for the identification of some of PI3K's activators and effectors. This was accompanied by the discovery that PI3K acts as a retroviral oncogene. The third decade was known for the establishment of the direct involvement of PI3K in cancer, demonstrated by the identification of cancer-specific mutations. Efforts to target PI3K were on the rise from that moment on, accompanied by the first clinical trials for PI3K inhibitor therapies. In the fourth decade of research, PI3K-based cancer drugs will continue to emerge, as will new knowledge regarding other uncovered functions of this protein and pathway.

The Importance of Being Non-Defective: A Mini Review Dedicated to the Memory of Jan Svoboda.

Jan Svoboda triggered investigations on non-defective avian sarcoma viruses. These viruses were a critical factor in the genetic understanding of retroviruses. They provided the single and unique access to the field and facilitated the discovery of the first oncogene src and of the cellular origin of retroviral oncogenes. They continue to be of importance as singularly effective expression vectors that have provided insights into the molecular functions of numerous oncogenes. Combined with the contributions to the validation of the provirus hypothesis, Jan Svoboda’s investigations of non-defective avian sarcoma viruses have shaped a large and important part of retrovirology.

Retraction for Fusco et al., “One- and Two-Step Transformations of Rat Thyroid Epithelial Cells by Retroviral Oncogenes”

Retraction for Fusco et al., “One- and Two-Step Transformations of Rat Thyroid Epithelial Cells by Retroviral Oncogenes”

Thyroid Hormone Receptors: Several Players for One Hormone and Multiple Functions.

Thyroid hormone receptors (TRs) were cloned based on their homology with the retroviral oncogene v-ERBA. In Vertebrates two genes, THRA and THRB, encode respectively many isotypes and isoforms of receptors TRα and TRβ, resulting from alternative splicing and/or internal transcription start sites. We present here a wide overview of this diversity and of their mechanisms of action as transcription regulators, as well as alternative actions through cytoplasmic signaling.

Highly efficient cellular cloning using Ferro-core Micropallet Arrays

Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the “ferro-core micropallet array”, to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest. Using transfected (retroviral oncogene or fluorescent reporter construct) rat 208 F cells, we demonstrated the capacity to isolate and expand pure populations of genetically manipulated cells via laser release and magnetic recovery of single micropallets carrying adherent microcolonies derived from single cells. This platform can be broadly applied to biological research, across the spectrum of molecular biology to cellular biology, involving fields such as cancer, developmental, and stem cell biology. The ferro-core micropallet array platform provides significant advantages over alternative sorting and cloning methods by eliminating the necessity for repetitive purification steps and increasing throughput by dramatically shortening the time to obtain clonally expanded cell colonies.

Q&A: Michael Bishop

Michael Bishop and Harold Varmus proved that genetic changes could drive the formation of tumours. They were awarded the 1989 Nobel prize in Physiology or Medicine for discovering the origin of retroviral oncogenes. Bishop — now director of the GW Hooper Foundation at the University of California, San Francisco — tells Kipp Weiskopf about 40 years in cancer research.

Duplication and maintenance of the Myb genes of vertebrate animals

SummaryGene duplication is an important means of generating new genes. The major mechanisms by which duplicated genes are preserved in the face of purifying selection are thought to be neofunctionalization, subfunctionalization, and increased gene dosage. However, very few duplicated gene families in vertebrate species have been analyzed by functional tests in vivo. We have therefore examined the three vertebrate Myb genes (c-Myb, A-Myb, and B-Myb) by cytogenetic map analysis, by sequence analysis, and by ectopic expression in Drosophila. We provide evidence that the vertebrate Myb genes arose by two rounds of regional genomic duplication. We found that ubiquitous expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, was lethal in Drosophila. Expression of any of these genes during early larval eye development was well tolerated. However, expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, during late larval eye development caused drastic alterations in adult eye morphology. Mosaic analysis implied that this eye phenotype was cell-autonomous. Interestingly, some of the eye phenotypes caused by the retroviral v-Myb oncogene and the normal c-Myb proto-oncogene from which v-Myb arose were quite distinct. Finally, we found that post-translational modifications of c-Myb by the GSK-3 protein kinase and by the Ubc9 SUMO-conjugating enzyme that normally occur in vertebrate cells can modify the eye phenotype caused by c-Myb in Drosophila. These results support a model in which the three Myb genes of vertebrates arose by two sequential duplications. The first duplication was followed by a subfunctionalization of gene expression, then neofunctionalization of protein function to yield a c/A-Myb progenitor. The duplication of this progenitor was followed by subfunctionalization of gene expression to give rise to tissue-specific c-Myb and A-Myb genes.

Retroviral oncogenes: a historical primer

Retroviruses are the original source of oncogenes. The discovery and characterization of these genes was made possible by the introduction of quantitative cell biological and molecular techniques for the study of tumour viruses. Key features of all retroviral oncogenes were first identified in src, the oncogene of Rous sarcoma virus. These include non-involvement in viral replication, coding for a single protein and cellular origin. The MYC, RAS and ERBB oncogenes quickly followed SRC, and these together with PI3K are now recognized as crucial driving forces in human cancer.

Introduction: Crk Signaling and Cancer- The First 25 Years

Introduction: Crk Signaling and Cancer- The First 25 Years

A conversation with Harold Varmus

For the first of our series Conversations with Giants in Medicine, we spoke with Harold Varmus, who has been the director of the National Cancer Institute since July 2010 (Figure ​(Figure1).1). He has previously served as director of the National Institutes of Health (1993–1999) and as president of the Memorial Sloan-Kettering Cancer Center (2000–2010). Among his many awards, Varmus was awarded the 1989 Nobel Prize in Physiology or Medicine together with J. Michael Bishop in recognition of their discovery of the cellular origin of retroviral oncogenes. The full interview can be seen on the JCI website, http://www.jci.org/kiosk/cgm.

The contrasting oncogenic and tumor suppressor roles of FES

The FES gene was first discovered as a protein-tyrosine kinase-encoding retroviral oncogene. The ability of v-FES to transform cells in vitro and initiate cancer in vivo has been established by cell culture, engraftment and transgenic mouse studies. The corresponding cellular c-FES proto-oncogene encodes a cytoplasmic FES protein-tyrosine kinase with restrained catalytic activity relative to its retrovirally encoded homologs. These observations have stimulated a search for mutations or inappropriate expression of c-FES in human cancers and research aimed at understanding the functions of the FES kinase and its potential involvement in cancer and other diseases. Paradoxically, although first identified as an oncogene, genetic evidence has also implicated c-fes as a potential tumor suppressor. This review will describe observations from basic and translational research which shapes our current understanding of the physiological, cellular and molecular functions of the FES protein-tyrosine kinase and its potential roles in tumorigenesis. We also propose a model to reconcile the conflicting oncogenic and tumor suppressor roles of c-FES in tumorigenesis.