Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

CST6 (cystatin E/M) Daniel Keppler Department of Biological Sciences, College of Pharmacy, Touro University of California, 1310 Johnson Lane, Mare Island, Vallejo, CA 94592, USA (DK)

Published in Atlas Database: July 2009 Online updated version : http://AtlasGeneticsOncology.org/Genes/CST6ID40178ch11q13.html DOI: 10.4267/2042/44772 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2010 Atlas of Genetics and Cytogenetics in Oncology and Haematology

in the 5' upstream region by an inverted, 290-bp Alu- Identity Sx(Sq) repeat. Other names: Cystatin-6; Cystatin-E; Cystatin M; Description Cystatin E/M Like most cystatin , the human CST6 is HGNC (Hugo): CST6 organized into three exons separated by two introns. Location: 11q13.1 Exon-1 is 294-bp long, contains the 5'-untranslated region (5'-UTR) and the starting ATG codon of the Local order: The human CST6 gene is located on the coding sequence. Exon-2 is 126-bp long. Exon-3 is long arm of 11 at 11q13.1. It corresponds 188-bp long, contains a TGA stop codon, the 3'-UTR as to a total DNA sequence of about 1,515 bp. Most other well as a typical AATAAA polyadenylation signal human cystatin genes (i.e., the genes for CST1 to CST9 followed by 20 bp. Intron-1 and intron-2 are 541- and and CST11) cluster on chromosome 20p11. 365-bp in length, respectively. Note Misleading annotations: Transcription -CSTB or CSTb (is a different cystatin gene) The human CST6 gene is transcribed into a single -Yeast CST6 (is an unrelated gene encoding a yeast mRNA species of about 607 nucleotides (nt). There are transcription factor) no alternate transcript species. The transcript is -Mouse cystatin E1 (mouse CRES-like) composed of a 5'-UTR of 53 nt, a coding sequence of -Mouse cystatin E2 (mouse testatin-like) 447 nt, and a 3'-UTR of 107 nt. A palyndromic structure located some 360 nt downstream of the AUG DNA/RNA initiation codon (or 26 codons upstream of the TGA stop codon) seems to be responsible for some sequence Note variation in that region. Indeed, several expressed The human CST6 gene is a tiny gene. Together with its sequence tags (ESTs) differ primarily if not solely in basic promoter, it spans about 2,500 bp and is flanked that region of the mRNA sequence.

Figure 1: In the above diagram are represented the various genes flanking the human CST6 gene. More information on these genes can be found at: Gene.

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Figure 2: Structure of the human CST6 gene. Exon-1 contains the 5'-UTR (in blue) and the starting ATG codon of the coding sequence (in magenta). Exon-3 contains a TGA stop codon and the 3'-UTR (in blue). More information on the CST6 gene organization can be found at: Entrez Gene.

Transcription from the CST6 gene promoter seems to Description be both constitutive and regulated. Numerous potential The three-dimensional organization of Cst6 (assuming SP1 binding sites (TESS/TransFac database v4.0) in the it is similar to that of chicken egg white cystatin shown CST6 promoter may account for a low to moderate in figure 4) is that of a compact five-pleated beta-sheet basal promoter activity in many tissues. that partially wraps around a central alpha-helix. It is High expression occurs only in a few tissues such as not clear what role glycosylation of residue N the skin, placenta, ovary, pancreas and the lungs. A 137 fulfills. Perhaps, N-glycosylation promotes binding of quite widespread expression of CST6 is also supported the to cells and entry into the by data extracted from gene expression libraries (GEO, endosomal/lysosomal system where Cst6 can interact GeneNote, GNF Symatlas, CGAP, EST, SAGE, and with target proteases. UniGene eNortherns). However, there are some conflicting data in the Expression literature suggesting that the CST6 mRNA is expressed Cst6 is a cell-secreted protein. In vitro, the majority (> in a tissue-specific manner mainly if not exclusively in 95%) of the protein accumulates in the media the skin. conditioned by the cells. In cells that overexpress Cst6, Expression from the human CST6 gene is prominent labeling of the Golgi apparatus can be seen epigenetically silenced in several tumor types (see using indirect immunofluorescence cytochemistry. below). The 5'-end of the CST6 gene including exon-1 has an unusually high ( ≥ 70%) content in G and C Localisation nucleotides. As a matter of fact, a typical CpG island In the human skin, where localisation of Cst6 has been spans across the transcription start site (bp +1) from bp most carefully explored, the protein is detected in the -186 to bp +320 and encompasses all of exon-1. Not stratum granulosum of the epidermis, in the outer root surprisingly, treatment of tumor cells by histone sheet of hair follicles, in the secretory coil epithelium deacetylase or DNA methyltransferase inhibitors of sweat glands, and in the inner, mature cells of results in 're-expression' of CST6 at levels similar to sebaceous glands. those seen in the normal or benign counterparts. Function The unusual GC content (~ 80%) of the 5'-UTR of the mRNA suggests that CST6 expression might also be Protease Inhibitor Function: The most widely regulated at the translational level by eIF-4E. accepted function of cystatins is that of protease inhibitors. The name 'cystatin' further reminds us that these endogenous protease inhibitors target cysteine No have been identified. proteases. In contrast to metallo- and serine proteases that are mostly secreted proteases, most cysteine Protein proteases are confined within cells where optimal pH and redox conditions favor their enzymatic activity. Note Thus, the majority of intracellular cysteine proteases The CST6 gene product, Cst6, is a typical secretory are inactivated by oxidizing conditions outside the protein. It is synthesized as a preprotein with a patent cells. Nevertheless, it is believed that cystatins inhibit N-terminal signal sequence. The protein is translocated cysteine proteases much faster than do oxidizing into the rough endoplasmic reticulum where about 30- conditions and, thereby, prevent excessive tissue 50% of the nascent Cst6 polypeptides are N- damage during the release of lysosomal enzymes. glycosylated. Upon SDS-PAGE, Cst6 harvested from Among the various types of intracellular cysteine most cell secretions migrates as two major forms, a 14- proteases, cystatins seem to target preferentially kDa unglycosylated and a 17- to 18-kDa glycosylated endosomal/lysosomal cysteine proteases of the papain form. family, such as cathepsin B, cathepsin K/O2, cathepsin L, cathepsin L2/V and cathepsin S.

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Figure 3: The above diagram depicts the primary structure of the Cst6 precursor. The first 28 amino acids represent a canonical signal peptide. The mature and secreted Cst6 molecule (in blue) contains two disulfide bonds (-S-S-), one N-glycosylation site (N 137 -CHO), and two distinct binding sites for lysosomal cysteine proteases (purple and yellow boxes). The purple boxes represent the amino acids (RMVG, QLVAG and PW) that are involved in the binding and inhibition of the cathepsins B, K, L, L2/V or S. The yellow box represents the critical amino acid (N 64 ) for binding and inhibition of lysosomal Asn-endopeptidase (AEP or mammalian legumain). Figure 4: Typical crystal structure of a secretory cystatin. The coordinates for the crystal structure of chicken egg white cystatin (1CEW) were obtained from the PDB database. A 3D-model of the cystatin was then generated using SwissPDB-Viewer. The N- and C-termini of the protein are marked by 'N' and 'C', respectively. The two conserved disulfide bonds are highlighted in yellow. The amino acids that are part of the two distinct binding sites for lysosomal cysteine proteases are labeled by purple and yellow boxes as described in the legend to figure 3. N 64 and W 135 are particularly important in this regard and are highlighted in blue. The amino acid numbering refers to that of the Cst6 preprotein, i.e., the protein with a 28-amino acid signal peptide (not present).

Some cystatins such as Cst6 are double-headed Epithelial barrier function: One important function of inhibitors and have a second inhibitory site, i.e., N 64 in Cst6 seems to be in the terminal differentiation of figures 3 and 4 above. Via this alternate inhibitory site, stratified squamous epithelial cells and in the formation Cst6 is capable of binding and inhibiting legumain-type of cornified envelops. Indeed, ichq mice with a null cysteine proteases such as AEP/mammalian legumain. mutation in the cst6 gene develop neonatal Cystatins do not inhibit caspases and calpains seem to abnormalities in skin cornification and desquamation be regulated in a different manner. Little is known that resemble Harlequin ichthyoses in humans. about the inhibitory potential of cystatins towards other However, no alterations in the CST6 gene were found types of intracellular cysteine proteases. in the DNA of patients with Harlequin ichthyosis.

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In mice, the lack of Cst6 function leads to severe other cancers such as cancers of the breast, prostate, dehydration and neonatal lethality. Before serving as a brain, lung, cervix and melanocytes. In most tumor substrate to transglutaminases and being deposited into tissues, CST6 seems to be epigenetically silenced rather cornified cell envelops, Cst6 is believed to be important than deleted or mutated. However, in one case (see in fine-tuning the enzymatic activities of below) more profound alterations in the human CST6 endosomal/lysosomal cysteine proteases such as gene have been observed. cathepsin L, cathepsin L2/V and AEP/mammalian Cervical cancer: One out of 19 primary tumors revealed legumain. Deregulated activity of these proteases could homozygous deletion of exon-1 sequences. Six other lead to abnormal activation of transglutaminases and primary tumors exhibited point mutations in the CDS disorders in cornification. of the CST6 gene. Two of these mutations (M34T and L131F) occurred in proximity to the consensus binding Homology sites for cathepsins (figure 6) and resulted in CST6 Gene orthologs: diminished affinity of the mutant inhibitor for cathepsin L. UniGene Species Chromosome Homology Germinal ID No germ-line mutations have been detected. Human Hs.139389 11q13.1 100%/149 aa Pig Ssc.9061 2p16-17 78%/149 aa Implicated in Cow Bt.5468 29 75%/148 aa Cancer progression Dog Cfa.23670 18 71%/149 aa Loss of heterozygosity (LOH) affecting the locus Rat Rn.9609 1q43 70%/149 aa 11q13 is quite common in cancers. This locus indeed harbors several tumor or metastasis suppressor genes Mouse Mm.36816 19 A (4.0 cM) 69%/149 aa such as BAD, MEN1, BRMS1, RASGRP2, GSTP1 and Worm Cel.5518 V 13%/143 aa CST6. In a study using differential RNA display it was initially established that human breast cancer cell lines Mutations exhibited lack or reduced CST6 expression when compared to immortal or normal counterparts. CST6 Note was coined a novel candidate tumor suppressor gene In 2004, CST6 was coined as a novel candidate tumor for breast cancer on October 1st, 2004. suppressor gene for breast carcinoma. Since then, this gene has been identified as a tumor suppressor gene for

Figure 5: Degree of amino acid homology among human cystatin (in %). Cst3, cystatin C; Cst5, cystatin D; Cst6, cystatin E/M; Cst7, cystatin F/leukocystatin; Cst4, cystatin S; Cst2, cystatin SA; Cst1, cystatinSN; and Cst8, CRES.

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Figure 6: This diagram depicts locations of six point mutations and one deletion affecting the CST6 gene that have been observed in cancer specimens. Amino acid numbering refers to the precystatin sequence as for figure 3.

Since then, several groups have reported on the lack or 10AT and MCF-12A, respectively). Treatment of diminished expression of CST6 in various cancer types CST6-negative tumor cells by the histone deacetylase (listed below). However, some groups also observed overexpression of CST6 in select cancer types (listed inhibitor Trichostatin A (TSA) or the DNA below). One of the challenges in current research on methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5- CST6 is to define the proteases targeted by CST6 and Aza) results in 're-expression' of CST6 at levels similar their precise role in the progression of the disease. to those seen in the normal or benign counterparts. Cancer types with diminished CST6 Overexpression of CST6 in breast cancer cells (MDA- MB-435S and T-47D) is associated with diminished expression tumor cell colony formation, proliferation, migration, Breast cancer Matrigel invasion and orthotopic tumor growth in scid Note mice. Using various approaches, several groups have Prostate cancer independently established that the human CST6 gene Note promoter is epigenetically silenced in breast carcinomas when compared to normal breast tissue. In In a study of matched pairs of normal/cancer one study, 24/40 (60%) breast carcinomas exhibited tissues, loss of CST6 expression was observed in 18/20 CST6 promoter hypermethylation as compared to 7/28 (90%) prostate cancers. Similarly, only 6% of prostate (25%) normal breast tissue samples. In another study, cancers exhibited strong Cst6 immunohistochemical 25/45 (56%) of primary tumors and 17/20 (85%) of staining as compared to 63% of normal tissues. lymph node metastases expressed reduced levels of Among prostate cancer cell lines, RWPE-1 and DU- CST6 when compared to normal breast tissues. CST6 145 express high and moderate levels of CST6, promoter hypermethylation could be demonstrated in respectively, whereas LNCaP, PC-3 and PC-3M 3/11 (27%) primary tumors and 8/12 (67%) lymph express little to no CST6. Treatment with TSA leads to node metastases. In 35% of neoplastic lesions, no strong upregulation of CST6 expression in all three cell association could be established between the loss of lines. In contrast, treatment with 5-Aza up to five days CST6 expression and promoter methylation. This had no effect. Further studies using methylation- suggests that besides promoter hypermethylation other specific PCR showed that prostate cancer cell lines and (structural or regulatory) mechanisms might operate to tissues had lower promoter methylation than normal prevent CST6 expression in cancer cells. tissues. DNA hypermethylation of the CST6 promoter Most established breast cancer cell lines also exhibited does therefore not account for the silencing of CST6 little or no CST6 expression (21MT-1, MCF-7, T-47D, expression in prostate cancer. Instead, histone ZR-75-1, Hs578T, SK-BR-3, MDA-MB-157, MDA- deacetylation and chromatin remodeling seem to be MB-361, MDA-MB-435S, MDA-MB-436, MDA-MB- responsible for diminished CST6 expression. 453, BT-474 and BT-549). Some established breast Similar to breast cancer cells, forced expression of cancer cell lines expressed moderate levels of CST6 CST6 in prostate cancer cells (PC-3) leads to (MDA-MB-231, MDA-MB-415 and MDA-MB-468) diminished tumor cell proliferation and Matrigel and only few (21PT, 21NT, 21MT-2 and BT-20) invasion. In addition, overexpression of CST6 also expressed levels of CST6 similar to normal or immortal selectively reduces expression of the target enzyme, counterparts (70N and 80N or 76N, MCF-10A, MCF- cathepsin B. Conversely, silencing of CST6 expression

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in a CST6-positive prostate cancer cell line (RWPE-1) target protease, cathepsin L, possibly explaining the leads to the exact opposite results from overexpression. reduced growth of the CST6 overexpressing cells. In mice, orthotopic injection of PC-3 cells Head and neck squamous cell overexpressing CST6 resulted in considerably smaller tumors when compared to vector controls. The CST6 carcinoma (HNSCC) tumors expressed reduced levels of cathepsin B. Note Lung cancer Comparison of the gene expression profiles (HuFL6800) of two matched pairs of primary and Note metastatic human oropharyngeal SCC cell lines (MDA- Two groups have recently reported on the epigenetic 686TU and LN) revealed relative overexpression of silencing of the CST6 gene in non-small cell lung CST6 in the metastatic cell line. Immuno-cytochemical cancer (NSCLC) using genome-wide expression analysis further showed that overexpression of CST6 in profiling. In one study, 2/5 (40%) primary tumors and the metastatic cell line was not homogenous. Instead, 1/5 (20%) normal lung tissues exhibited CST6 small clusters of cells overexpressed the protein promoter methylation. In the other study, the numbers whereas the majority of cells expressed little or no were respectively 10/19 (53%) and 2/15 (13%). CST6. Further studies using RNA interference NSCLC cell lines that express little or no CST6 are the indicated that loss of CST6 expression in MDA-686LN following: A-427, A-549, NCI-H23, NCI-H522, NCI- promoted proliferation of the cells and Matrigel H1299 and NCI-H460. Three cell lines expressed invasion. moderate to high levels of CST6 (NCI-H322, NCI- In another study, human SCC-25 cells were treated H358 and NCI-H292). In all nine above cell lines, with the vitamin D3 analog EB1089 for various times CST6 expression could be increased to normal levels and the effect of this drug treatment on gene expression by a combined treatment of the cells with TSA and 5- analyzed using HuGene FL oligo microarrays. In this Aza. study, CST6 expression was found to increase > 30- Overexpression of CST6 in lung adenocarcinoma A- fold over a 24-hr period. Overall, EB1089 treatment 549 cells resulted in a > 50% reduction in colony reversed the malignant phenotype of SCC-25 cells and formation in vitro compared to vector controls. induced keratinocytic differentiation. Cervical cancer Brain cancer Note Note One study recently reported on the lack of CST6 One study reported on downregulation of CST6 expression in 9/11 (82%) primary squamous cell expression in 15/17 (88%) brain tumors, which carcinomas of the cervix, but expression of the gene in included 7/9 (78%) multiform glioblastomas (MG). 5/5 (100%) normal cervical tissues as well as in normal Moreover, MSP analysis demonstrated CST6 promoter lung, thyroid, kidney, brain, ovary, uterus, smooth methylation in 17/30 (57%) brain tumors. These latter muscle and connective tissues. Two out of 11 (18%) included 14/19 (74%) MGs. In comparison to brain primary tumors (one of which being an tumors, normal brain tissue exhibited only 6% CST6 adenocarcinoma) expressed low levels of CST6, which promoter methylation. might be due to contamination of the tumor material by CST6 expression and methylation status was also adjacent normal tissue. analyzed in six glioblastoma cell lines: LN-229, LN-18, Cervical cancer cell lines such as HeLa (D98/AH-2), T98G, DBTRG-05MG, U-87MG and U-118MG. All C41, SiHa, Caski, HT3 and C33A all lack expression of six cell lines expressed little or no CST6. In addition, CST6. Treatment of tumor cells by 5-Aza and/or TSA all cell lines had quite homogenously hypermethylated results in 're-expression' of CST6 at levels similar to CST6 promoters. Re-expression of CST6 could be those seen in normal tissues. Similar to the situation in triggered with 5-Aza alone. prostate cancer cells, some cell lines (SiHa and HT3) Transfection of T98G, LN-229 and U-87MG cells with respond only to TSA treatment. Caski, C33A and C41 a mammalian CST6 expression vector resulted in a cells exhibit both unmethylated and hypermethylated modest (20-25%) suppression of T98G and LN-229 CST6 promoters whereas HeLa cells has cell growth when compared to vector controls. Forced homogenously hypermethylated CST6 promoters. expression of CST6 in U-87MG cells had no effect on Overexpression of CST6 in HeLa and SiHa cells leads their capacity to form colonies and proliferate. in both cases to a reduction in the number and size of In conclusion, CST6-mediated suppression of tumor colonies forming in soft agar and in cell proliferation. cell growth seems to be most pronounced in cells of Another consequence of the forced expression of CST6 epithelial origin, i.e., in cells developing multiple cell- in HeLa cells is a reduction in intracellular levels of the to-cell communications and elaborating a basement membrane.

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Cancer types with increased CST6 (V600E) mutational status. CST6 expression was also expression associated with PTC lymph node metastasis. Ovarian cancer (OvCA) Squamous cell carcinoma of the skin Note Note In order to better define the molecular profiles of the Squamous cell carcinoma (SCC) of the skin versus four major histological types of OvCAs (clear cell, psoriasis. mucinous, endometrioid, and serous), a microarray CST6 is highly expressed in the normal human skin, analysis was performed on 113 human specimens. which might explain why no further increase in Expression of CST6 was found to be on average 3.8- expression could be detected in SCC. However, a five- fold higher in clear cell OvCAs when compared to to six-fold differential expression of CST6 was other histological types. It is interesting to note here observed when SCC was compared to psoriatic skin. that more than one-half of clear cell OvCAs do not Differential expression of CST6 was accompanied by a exhibit tumor invasion at presentation. similar differential expression of one of its target proteases, cathepsin L2/V. Breakpoints Pancreatic cancer Note Note A 300-kb region flanked by the markers D11S4908 and CST6 was identified as an upregulated gene in several D11S5023 and harboring the CST6 gene has been genome-wide expression studies. One study used identified as the minimal tumor deletion on 11q13 in microarray analysis to profile gene expression in cervical cancer cell lines and primary cervical tumors. pancreatic adenocarcinomas (T=10), pancreatic cancer This region was reported to contain a high density of cell lines (C=7), chronic pancreatitis (P=5) and normal DNA repeats rendering it fragile and prone to potential pancreas (N=5). According to this study, CST6 levels DNA breaks and carcinogenesis. A rare fragile site change 20-, 20- and 24-fold in T/N, T/P and C/N, FRA11A overlaps indeed with this region. respectively. In another study using a similar approach (oligo microarray) the T/N ratio was found to be 4.4- fold and upregulation of CST6 was not observed using References other platforms such as SAGE or cDNA-based Ni J, Abrahamson M, Zhang M, Fernandez MA, Grubb A, Su J, microarrays. Instead, among six genes that were Yu GL, Li Y, Parmelee D, Xing L, Coleman TA, Gentz S, consistently overexpressed across all three platforms Thotakura R, Nguyen N, Hesselberg M, Gentz R. Cystatin E is a novel human cysteine proteinase inhibitor with structural was one of the major CST6 targets, cathepsin L2/V. resemblance to family 2 cystatins. J Biol Chem. 1997 Apr In yet another study using a cDNA microarray, CST6 18;272(16):10853-8 was found to be overexpressed in 18 microdissected Sotiropoulou G, Anisowicz A, Sager R. Identification, cloning, pancreatic ductal adenocarcinomas (PDAC) when and characterization of cystatin M, a novel cysteine proteinase compared to normal ductal epithelial cells. Subsequent inhibitor, down-regulated in breast cancer. J Biol Chem. 1997 silencing of CST6 expression in a PDAC cell line (PK- Jan 10;272(2):903-10 59) reduced colony formation and cell proliferation. Stenman G, Aström AK, Röijer E, Sotiropoulou G, Zhang M, Conversely, overexpression of CST6 in a CST6- Sager R. Assignment of a novel cysteine proteinase inhibitor negative PDAC cell line (KLM-1) promoted tumor (CST6) to 11q13 by fluorescence in situ hybridization. Cytogenet Cell Genet. 1997;76(1-2):45-6 growth in nude mice. Likewise, addition of recombinant human CST6 to the growth medium of Brillard-Bourdet M, Nguyên V, Ferrer-di Martino M, Gauthier F, Moreau T. Purification and characterization of a new cystatin KLM-1 cells promoted their proliferation in a dose- inhibitor from Taiwan cobra (Naja naja atra) venom. Biochem dependent manner. Engineered CST6 variants lacking J. 1998 Apr 1;331 ( Pt 1):239-44 either N-glycosylation (N137D, figure 3) or with an Alvarez-Fernandez M, Barrett AJ, Gerhartz B, Dando PM, Ni J, altered protease binding site (deletion of MVG38, Abrahamson M. Inhibition of mammalian legumain by some figures 3 4) did not have any effect on cell proliferation cystatins is due to a novel second reactive site. J Biol Chem. suggesting that both N-glycosylation and protease 1999 Jul 2;274(27):19195-203 specificity are required for oncogenic activity of CST6. Zeeuwen PL, Van Vlijmen-Willems IM, Jansen BJ, Sotiropoulou G, Curfs JH, Meis JF, Janssen JJ, Van Ruissen Thyroid cancer F, Schalkwijk J. Cystatin M/E expression is restricted to Note differentiated epidermal keratinocytes and sweat glands: a new Initial immunohistochemical studies found positive skin-specific proteinase inhibitor that is a target for cross- linking by transglutaminase. J Invest Dermatol. 2001 staining for CST6 in 80% (8/10) of papillary thyroid May;116(5):693-701 carcinomas (PTC) and 73% (11/15) of benign thyroid Hong J, Yoshida K, Rosner MR. Characterization of a cysteine lesions. Independent studies established a strong proteinase inhibitor induced during neuronal cell differentiation. correlation between CST6 expression, PTC and BRAF J Neurochem. 2002 Jun;81(5):922-34

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