Genetic amplification of IQGAP1 in cancer

#This invention is related to the area of cancer prognosis and therapeutics. In particular, it relates to a gene that is amplified in human cancers

#The human IQGAP protein family comprises three members, which, as scaffold proteins, play important roles in the regulation/modulation of cytoskeletal architecture and cell adhesion through binding to various effectors/regulators (1-4). Among these, IQGAP1 has been best characterized (3-6). This protein was initially identified in human osteosarcoma cells and consists of a 1,657-amino acid polypeptide with significant similarity to the Ras-related GTPase-activating (RasGAP) proteins (7). A unique structural feature of IQGAP proteins is their IQ domain, which contains about 25 amino acids and is found in diverse families of calmodulin-binding proteins. This motif is capable of effecting on intracellular Ca2+ signaling and a wide range of cellular activities. Some previous studies showed that IQGAP1 might be able to modulate the Ras→Raf→MEK→MAP kinase/ERK pathway (MAP kinase pathway) in certain cells, but it can also function independently of this mechanism (8-10). Overexpression of IQGAP1 was seen in certain cancers, suggesting a role of this molecule in human tumorigenesis (3,4,11-13). IQGAP3 may potentially also have such a role as it was shown to be involved in cell proliferation (2). IQGAP2 has been less well studied and may function as a tumor suppressor (4).

#Follicular epithelial cell-derived thyroid cancer is a common endocrine malignancy (14). This cancer can be histologically classified into papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), and anaplastic thyroid cancer (ATC). PTC can be further classified into several variants or subtypes, including mainly conventional PTC (CPTC), follicular variant PTC (FVPTC), and tall cell PTC (TCPTC). A striking genetic alteration in PTC is the BRAF mutation, which plays an important oncogenic role through activation of the MAP kinase pathway (15-17). ATC is a rare but rapidly lethal cancer with aggressive invasion and metastasis. PTC and FTC are both differentiated thyroid cancers with a relatively indolent clinical course, but FTC is generally more invasive and aggressive than PTC except for certain types, such as TCPTC, which is also relatively aggressive (18, 19). FTC and FVPTC tend to display invasive behaviors, such as vascular invasion (20). It is not known whether IQGAP proteins play any role in thyroid tumorigenesis or whether it determines aggressiveness our outcome in other cancers. There is a continuing need in the art to obtain better tools for diagnosing, prognosing, and treating human cancers.

#FIGS. 1A-1C show immunohistostaining (IHS) of IQGAP1 in thyroid cancer. Presented are representative tumor samples with various IQGAP1 copy numbers (CNs) and corresponding IHS of the IQGAP1 protein. A brown color of staining using anti-IQGAP1 antibodies represented IQGAP1 protein. Cases A, B, and C represent IHS scores I, II, and III, respectively, as defined in Example 1. Corresponding IQGAP1 CNs are indicated in the brackets.

#FIGS. 2A-2B show effects of shRNA knockdown of IQGAP1 or IQGAP3 on the invasion of thyroid cancer cells. SW1736, KAT18, and FTC133 cells, at a density of 2.5×104 cells/well, were seeded on Matrigel invasion chamber plates and cultured in routine medium for 22 hours. Invasive cells that penetrated through pores and migrated to the underside of the membrane were stained by crystal violet and photographed under a microscope. FIG. 2A shows representative results on the invasion assay of three thyroid cancer cells. FIG. 2 B is a histogram plots of the number of invading cells corresponding to FIG. 2A for each cell type. “(C)” denotes cell transfection with shRNA containing scrambled nucleotides; “(S)” denotes cell transfection with specific shRNA to knock down IQGAP1 or IQGAP3. **, P<0.01.

#FIGS. 3A-3B. Effects of shRNA knockdown of IQGAP1 and IQGAP3 on anchorage-independent colony formation of thyroid cancer cells. SW1736 and FTC133 cells stably transfected with control shRNA or specific shRNA to knock down IQGAP1 or IQGAP3 were seeded in 6-well plates in 0.3% agar over a bottom layer of 0.6% agar and treated with routine medium plus 1.5 mg/mL puromycin. After 2-3 weeks, cell colony numbers were counted under microscope and cell colonies were photographed. FIG. 3A, representative results of colony formation of SW1736 and FTC133 cells. FIG. 3B, histogram plots of colony numbers corresponding to A for each cell type. Only cell colonies containing more than 50 cells were counted. KAT18 cells did not grow on soft agar. “(C)”, cell transfection with shRNA containing scrambled nucleotides; “(S)”, cell transfection with specific shRNA to knock down IQGAP1 or IQGAP3. *, P<0.05; **, P<0.01.

#The inventor has investigated the oncogenic role of IQGAP1 in cancer and its potential as a prognostic marker and therapeutic target in cancer. A genetic copy gain of IQGAP1 was found to be associated with increased invasiveness and aggressiveness of thyroid cancer. IQGAP1 genetic copy gain was preferentially seen in aggressive types of thyroid cancer and highly associated with extra-thyroidal and vascular invasion. In BRAF mutation-positive papillary thyroid cancer (PTC), IQGAP1 copy gain was associated with a tumor recurrence rate of 60%. In vitro knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion. The clinical implications of these results are at least two: (i) IQGAP1 copy gain can be used to predict invasiveness and aggressiveness of thyroid cancer and, when coexisting with BRAF mutation is a particularly powerful predictor for cancer recurrence; (ii) IQGAP1 represents a therapeutic target for cancers, including thyroid cancers.

#Copy number gain may be assessed by any means known in the art. These include without limitation, FISH (fluorescent in situ hybridization), In situ hybridization, Molecular cytogenetics, Virtual Karyotype, Happy mapping, Single Molecule RNA, Fiber FISH, Q-FISH, Flow-FISH, and digital karyotyping. Variations and alternatives to these techniques can be used. The gain can be measured using either a reference gene in the same tissue as the test tissue or using the IQGAP1 gene in a reference (normal) tissue. The amplification that has been observed has been relatively modest, with copy numbers of greater than 2, greater than 3, or greater than 4, although larger gains are possible, such as greater than 5, 6, 7, 8, 9, or 10 copies.

#IQGAP1 is the official symbol for the gene named IQ motif containing GTPase activating protein 1 in Homo sapiens. Other names for this gene include HUMORFA01, KIAA0051, SARI, p195, RasGAP-like with IQ motifs; and ras GTPase-activating-like protein IQGAP1. The gene is located on chromosome 15 at location: 15q26.1 in the human genome. Its annotation is chromosome 15, NC_000015.9 (90931473 . . . 91045475). The protein contains four IQ domains, one calponin homology domain, one Ras-GAP domain and one WW domain. It interacts with components of the cytoskeleton, with cell adhesion molecules, and with several signaling molecules to regulate cell morphology and motility. See NP_003861.