Prof. Dan Canaani lab.-Preface
Isolation and identification of human DNA repair genes via expression cloning with a selectable transfected clone cDNA library
We initiated this project by immortalizing with origin-defective SV40 DNA, a xeroderma pigmentosum group C cells; a genetic group not represented until then by an immortal cell line (Canaani et al., 1986; Naiman et al. 1989). In a pioneering attempt to clone human genes using functional complementation (UV resistance) as a selection to said XP-C cells transfected by a human cDNA library (Teitz et al., 1987). We isolated two human genes: the regulatory subunit of casein kinase 2 (Teitz et al., 1990a), and a novel human gene that we named UV Resistance Associated Gene (UVRAG, Perelman 1987). Noteworthy, The XP-C gene was identified only 5 years later by Legerski (1992) who have used a different, full-length cDNA library.
We have predicted that that CK2 modulates the DNA damage response by enhancing the G2/M checkpoint control (Teitz et al., 1990a), a claim verified by Glover and Hartwell (1995-1998), as well as by us (Dotan et al., 2001). We have also shown some of the complex regulation of CK2 subunits while designing an autocatalytic conditional mammalian system (Dotan et al., 1995). Together with Yang-Feng, we chromosomally mapped all three CK2 subunit genes (Yang-Feng et al., 1990, 1991 & 1994).
As for the novel gene (UVRAG) which we have isolated and characterized (Teitz et al., 1990b; Perelman 1997), we have predicted that it will turn out to be a tumor suppressor gene (PCT on potential usages of UVRAG, applied on 1995 and approved 1999). Indeed, our collaboration with Jung, then at Harvard Univ., has shown that this gene is a colon tumor suppressor gene that is essential for autophagy (Liang et al., 2006).
Developing the methodology of synthetic lethality screens in cultured human cells and mouse embryo fibroblasts
The genetic synthetic lethality screening is one of the most powerful methods for identification of functional interactions between genes in yeast. As of 1995, I desired to develop such a method in human cells. I planned the technology to employ both an immortalized human cell line, deficient in a gene of interest, and a complementing episomal survival plasmid expressing the gene of interest. The episomal plasmid is tagged by one of a novel double-label fluorescence system, while the host cells are marked by a chromosomally integrated fluorescent gene. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors should prevent the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells are grown in micro titer plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, or identification of gene targets, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. However, fluorescent proteins (GFP) fit for double-label became available only by June 1977 (Packard Instruments), enabling us to initiate the project. By 2001 we published the generation of a chemical synthetic lethality system in human cells (Simons 2001a), and a genetic synthetic lethality system (Simons 2001b). We then constructed such chemical- (Einav et al., 2003) and genetic-synthetic lethality screening systems (Einav et al., 2005) in mouse embryo fibroblasts (MEFs). The reasoning being the availability of a large number of immortalized MEFs derived off knockout mice, thus constituting a potential rich source of cell recipients for genetic synthetic lethality screens. Initially our genetic suppressor elements were composed of truncated sense and short anti sense RNAs (Simons 2001b). However, with the discovery of the RNAi phenomenon we switched to the more effective shRNAs (Einav et al., 2005; Boettcher et al., 2010). The later involved collaboration with Hoheisel group, to improve the pooled RNAi based genetic synthetic lethality screens (Boettcher et al, 2010). Over these past recent years, I reviewed twice the progress in this cancer-related field, primarily from the experimental point of view (Canaani 2009; 2014).
Human SKAI1BC lncRNA: potential target for therapy of diverse cancers
My fascination with the discovery of thousands of human lncRNAs which may regulate the protein coding genes, have led me to attempt finding potential targets for triple-negative breast cancer therapy among lncRNAs.
As outlined at length in my "preliminary results" section, we have tested in a meticulous fashion (Tzadok et al., 2013; Aram et al., 2017) whether there are promoter-spanning anti-sense lncRNAs among ten epigenetically silenced human breast tumor suppressor/metastasis suppressor genes. We have identified only one such lncRNA whose expression emerges to be inversely related to the KAI1 mRNA expression and in direct relationship to the invasiveness level of human breast cancer derived cell lines. Importantly, KAI1 acts as a metastasis suppressor in at least 18 solid cancers, including breast. Knockdown of this KAI1 antisense lncRNA in the triple-negative breast cancer cell line MDA-MB-231 have led to increased KAI1 mRNA and protein expression, manifested in stronger adhesion to fibronectin, retardation of cell migration and reduced cell invasion in vitro. Thus we have named this novel lncRNA, SKAI1BC, standing for "Suppressor of KAI1 in Breast Cancer" (Aram et al., 2017). In this project, we took advantage of the phenomenon of nuclear RNAi, which we intensively characterized in collaboration with Shav-Tal's group (Avivi, 2017).
Broad-spectrum metastasis suppressing compounds and therapeutic uses thereof in human tumors
Despite recent advances in cancer therapy, still malignancy is responsible for 15% of human death. Among those patients having solid tumors, 90% die from metastatic disease.
Thus, effective prevention and suppression of metastasis is still an elusive goal. Previously, we have identified a novel human metastasis inducing lncRNA, which suppresses the KAI1/CD82 metastasis-suppressing gene, and is upregulated in TNBC and melanoma derived cell lines (Aram et al. 2017). Here we show identification of five compounds, belonging to two chemical groups, which inhibit at low concentration metastasis invasion and cell migration in culture without affecting cell proliferation. This was found in eight types of solid human cancers, out of the eight types tested; among which several of the most lethal and/or frequent human malignancies (Gottfried-Komlosh et al., in preparation). Moreover, based upon the mechanism of action of several of our compounds, coupled to epigenetic inactivation of KAI1/CD82 in 10 additional solid human cancers, there is a good chance to broaden the spectrum of human cancers affected by our compounds. Thus, the proposed compounds and/or their derivatives may identify a novel therapy for a broad-spectrum of the major human solid tumors.