The Role of Testin in Human Cancers

Testin is a protein with a molecular mass 47 kDa encoded by TES gene located on the fragile site FRA7G at 7q31.2 [6]. It is localized in the cytoplasm, along stress fibers and is recruited to focal adhesions [12]. The protein is composed of N-terminal PET (Prickle, Espinas, TES) and C-terminal three LIM (lin–11, isl-1, mec-3) domains [13]. At its COOH terminal (Fig. 1), the testin protein has three zinc-binding domain linked by two amino-acids spacer which play role in focal adhesion. LIM family proteins have been found to be a part of cytoskeleton [14]. They are responsible for protein-protein interactions coordinating signaling intracellular pathways [15, 16]. The N-terminal and C-terminal halves of the protein can interact with each other, hence hindering interaction with other cytoskeleton associated protein such as: zyxin, vasodilator stimulated protein (VASP), talin, nuclear actin related protein (Arp7A) and actin [17]. They are together acting as integrating partners in focal adhesion. Coutts et al. noticed that fibroblasts stably overexpressing TES have an increased ability to spread, are larger and contain increased numbers of actin protrusions [16]. Testin protein overexpression had effect on cells spreading potential not on percentage of cells spread. In view of described built, testin plays significant role in the cell adhesion, cell spreading and in the reorganization at the actin cytoskeleton [5, 18, 19].

Molecular studies characterized TES as tumor suppressor gene and reported its downregulation in many human malignancies [1‐3, 6‐8, 20]. In 2007 Boëda et al. presented for the first time interaction between LIM3 domain of testin protein and Mena protein which is key modulator of cellular migration [18]. Moreover, it was shown, that decreased expression of testin protein increased cell motility, decreased cell-cell contact and therefore have potential to be a marker of cancer metastasis [4, 21, 22].

TES encodes testin protein containing a PET domain at the NH2- terminus which is involved in actin stress fibers targeting, and three LIM ((lin-1, ils-1 and mec-3) domains (LIM1, LIM2, LIM3) at the COOH-terminus. One LIM domain contain loosely conserved cysteine-rich consensus sequence including two separate zinc fingers-F1. They are separated from each other by SPACER.

Cancers are the only investigated diseases with TES gene disruptions. It is silencing promotes cell proliferation, invasiveness ability and angiogenesis [23, 24]. Important ways of TES inactivation are mechanisms of loss of heterozygosity (LOH) and hypermethylation (HMT). In LOH one or two alleles of the same gene are lost, whereas in HMT occurs abnormal DNA methylation which may inactivate suppressor genes. This phenomena were described in almost every type of cancer. Predominantly, in performed studies decreased TES gene expression associated with HMT of CpG islands nor LOH of chromosome 7q31 was found [3, 19, 25]. In addition, methylation of TES promoter region was described in various tumor types. Tobias et al. showed methylation of the CpG islands at the 5′ end in many types of tested tumor-derived cell lines [26]. Tatarelli et al. found fully methylated TES promoter in 1/10 breast, 1/8 pancreatic and 9/18 leukemia cell lines [19]. According to Ma et al. methylation of CpG in the TES promoter inactivate gene. Moreover, it was revealed that treatment with 5-aza-2’deoxycytidine (DAC), inhibitor of DNA methyltransferase activity, switched completely methylated TES promoter into partially or even fully unmethylated region in gastric cancer cell lines [3]. Upregulation of TES gene expression after treatment with DAC in glioblastoma cells confirmed that HMT play significant role in TES regulation, being responsible for gene silencing [27]. Only one study presented contrary results. Han et al. indicated overexpression of TES in GTL-16 gastric cancer cell line [28]. High frequency of LOH was found at 7q31 region in primary gastric cancer, they identified D7S486 to be the most frequent LOH locus [3]. As it was anticipated, there is an evidence presence of LOH in 7q31.2 in many types of neoplasms, e.g. ovary, breast, colorectal, gastric, head and neck, prostate, thyroid, pancreatic and kidney cancer as well in leukemias [29‐37]. Ma et al. presented correlation between LOH presence and lack of testin protein expression in gastric cancer [1, 3]. Results are unequivocal as Chene et al. did not disclose such correlations in prostate cancer [4].

According to GDC Data Portal which analyzed 10,202 cases they identified 105 cases with different type of cancers (Fig. 2).

Analyzed patients was affected by 114 mutations of TES gene described in 21 projects. The largest number of cases was covered by endometrial carcinoma (42 cases), colorectal cancer (12) and breast cancer (7). During the process of transcription was identified missense, stop gained and frameshift mutations (Fig. 3).

The most frequent type of somatic mutation was substitution, described in 91 cases. Two authors cloned and described human TES gene. Tatarelli et al. determined that inactivation of TES is caused by methylation of CpG islands and revealed 3 missense mutation in 26 tumor cell lines [19]. In 2001 Tobias et al. showed also frequent occurrence of methylation of the CpG island at the 5-prime end in ovarian cancer and tumor-derived cell lines [26]. Research on TES gene revealed that TES gene may represent tumor supressor gene.

The datasets analysed in the current study are available on the in the Genomic Data Commons (GDC) Data Portal at https://​gdc.​cancer.​gov/​.