Identification of tumor suppressor genes using the approach of gene inactivation test

Epithelial cancers are the most prevalent and lethal diseases in the world. They cause more than 80% of all cancer deaths. Development of these tumors is a complex process involving more than 100 genes from different chromosomes. We know that human chromosome 3 (chr.3) contains many of these key tumor suppressor genes (TSGs), some of which are cancer specific and others are common for different cancer types.

The most widely used approach to designate a candidate gene as being a TSG is demonstration of inactivating mutations of this gene in tumor biopsies. Even such a simple requirement for mutational inactivation can be complicated in cases where expression dosage is critical, and in genes in which one allele is imprinted. In such cases, only functional approaches that can demonstrate tumor suppression activity for one of the candidate genes can help to solve the problem. Gene inactivation test (GIT) is a new functional test system for TSGs identification. This test is based on the functional inactivation of the analysed genes in contrast to existing tests based on growth suppression.

Our hypothesis was that TSG must be inactivated in growing tumors in experimental conditions as it happened in nature. This inactivation of a TSG can be achieved by mutation, deletion, methylation etc. To verify our hypothesis, known suppressor genes RB and p53 were built into the vectors that permitted tetracycline/doxycycline regulated expression of the cloned genes in cancer cell lines growing not only in vitro, but in vivo as well. These cell lines are tTA producing cell lines. Wild type but not mutated RB and p53 genes were deleted/inactivated during tumor growth in SCID mice. In contrast, a non-functional mutant RB and p53 gene were maintained.

Our group has tested approximately twenty candidate genes from 3p in SCLC lines U2020, ACC-LC5, prostate LNCaP and the RCC cell line KRC/Y using the GIT. These experiments led to the surprising conclusion that 3p contains at least 10 new TSGs. TSG may be a tissue specific gene and therefore, GIT can be effective only in some cell lines. As our research is focused on human 3p, we have collected cell lines with rearranged short arm of Chr.3, and a few control cell lines were selected as well. The panel of cell lines included 12 cancer cell lines. Our recent results showed that GIT could be very useful in functional analysis of new and well-known TSGs.

In summary, RB, P53, NPRL2/G21 RASSF1A, RASSF1C, RBSP3, SEMA3B, HYAL1, HYAL2 and CACNA2D2 demonstrated TSG activity in GIT at least in one tested cancer cell line. However, all mutated genes and wild type PL6, RhoA, 3PK, BLU, 101F6, MLH1 and others didn't show this activity in the tested cell line(s), although they resided in frequently affected 3p21.3 regions. Some of the newly discovered TSGs revealed unique features. For instance, HYAL1 and HYAL2 show growth inhibiting activity only in vivo. RASSF1A and RASSF1C are tissue-specific splice forms of the same TSGs. NPRL2 is likely a new mismatch repair gene responsible for cisplatin (one of the most widely used anti-cancer drug) action. NPRL2 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signalling and activation of apoptotic pathway. RBSP3 is the first known direct activator of RB by its dephosphorylation.

These new TSGs from AP20 and LUCA region are responsible for cell cycle control, mismatch repair, cell adhesion and angiogenesis and most likely cooperate with each other. The cloning of TSGs from Chr.3, and the comparative analysis of genes involved in the development of RCC, SCLC, and other cancers, will lead to a better understanding of the molecular mechanisms in carcinogenesis. It will improve the possibilities of early diagnosis and will allow more effective monitoring of treatment compared with present-day therapies.

List of scientific papers

I. Li J, Wang F, Protopopov A, Malyukova A, Kashuba V, Minna JD, Lerman MI, Klein G, Zabarovsky E (2004). "Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene." Oncogene 23(35): 5941-9
https://pubmed.ncbi.nlm.nih.gov/15208682

II. Li J, Wang F, Haraldson K, Protopopov A, Duh FM, Geil L, Kuzmin I, Minna JD, Stanbridge E, Braga E, Kashuba VI, Klein G, Lerman MI, Zabarovsky ER (2004). Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C. Cancer Res. 64(18): 6438-43.
https://pubmed.ncbi.nlm.nih.gov/15374952

III. Kashuba VI, Li J, Wang F, Senchenko VN, Protopopov A, Malyukova A, Kutsenko AS, Kadyrova E, Zabarovska VI, Muravenko OV, Zelenin AV, Kisselev LL, Kuzmin I, Minna JD, Winberg G, Ernberg I, Braga E, Lerman MI, Klein G, Zabarovsky ER (2004). RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies. Proc Natl Acad Sci U S A. 101(14): 4906-11. Epub 2004 Mar 29
https://pubmed.ncbi.nlm.nih.gov/15051889

IV. Wang F, Li J, Grigorieva E, Pavlova T, Lerman MI, Kashuba V, Klein G, Zabarovsky ER (2006). Gene inactivation test identfied HYAL1 and HYAL2 as tumor suppressor genes. [Manuscript]