Expression of Transcription Factor 
Cyclic AMP element modulator (CREM) is a transcriptional regulator protein that belongs to the cAMP response element–binding protein (CREB) transcription factor protein family. This is a family of basic domain-leucine zipper proteins with a capacity to bind to the cAMP response element (CRE), which is a sequence present in the regulatory region of a large number of target genes. , The CREM gene has several promoters that give rise to multiple isoforms. Different isoforms of CREM can promote or repress expression from the CRE. The gene product from its promoter P2 represses transcription from the CRE and is denoted as inducible cAMP early repressor (ICER). CREM protein is known to be highly expressed in the testis, where it is essential for spermatid development. , A few studies have found the functional roles of CREM in the central nervous system (CNS). CREM expression has been found to increase in the rat CNS after neuronal damage, proposing a role in CNS injury and repair. It has also been discovered to have a role in mediating impulsive behavior and addiction, as well as in regulating the spinal morphology and neuroplasticity. A few studies have linked altered CREM protein expression to cancer, more specifically to prostate and esophageal carcinoma. , Recently, the CREM gene has been found as a fusion gene partner in several types of tumors. The most commonly found CREM rearrangement is the EWS RNA binding protein 1 ( EWSR1 )- CREM fusion gene. It is recurrent in several types of neoplasia such as mesenchymal tumors, in hyalinizing clear cell carcinoma of salivary glands, and, most recently, in a malignant epithelioid neoplasm with predilection for mesothelial-lined cavities. – The EWSR1-CREM fusion genes encode fusion proteins with an N-terminal EWSR1 transactivation domain and a C-terminal DNA-binding domain of CREM. Detection of the fusion protein or neoexpression of the CREM protein could potentially serve as a surrogate for genetic testing, if the expression of wild-type CREM protein in tissues would be low. Furthermore, a basic description of CREM expression in tissues and cell types would be instrumental for studying the functional roles of CREM in both physiological and disease-associated processes. Despite this, the expression of CREM protein has not been studied using an antibody that targets the C-terminal DNA-binding domains of CREM, which are present in virtually all CREM isoforms. To bridge this gap in knowledge, we herein characterize the expression of CREM in human tissues, using publicly available transcriptome databases, and on protein level using immunohistochemistry (IHC) of tissue microarrays (TMAs) built for this purpose using an antibody targeting the C-terminal part of most CREM isoforms. With the expression pattern of CREM portrayed, we also test whether CREM IHC could be used as an indicator of the presence of EWSR1-CREM fusion gene in low-grade mucoepidermoid carcinoma (MEC). 

Cell Culture The commercially available human skin melanoma CHL-1, the human embryonic kidney HEK-293, and the human prostate carcinoma PC-3 were obtained from American Type Culture Collection (Manassas, VA). CHL-1 and HEK-293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 5 mM ultraglutamine, and 100 U/ml penicillin-streptomycin (Gibco; Carlsbad, CA), whereas PC-3 cells were cultured in RPMI medium with the same supplements. CREM Knockdown The CREM transcripts were knocked down in CHL-1, HEK-293, and PC-3 cells using 50 nM of human CREM small interfering RNA (siRNA) (sc-37700; Santa Cruz Biotechnology, Santa Cruz, CA) containing a pool of three to five target-specific 19–25 nucleotide sequences in length. A mix of three different non-targeting siRNAs (sc-37007, sc-44230, and sc-44231; Santa Cruz Biotechnology) was used as control. Cells were transfected using Dharmafect 4 transfection reagent (Dharmacon Research) according to the manufacturer’s instructions. The knockdown efficacy was examined 48 hr after transfection by Western blotting. The experiments were repeated at least 3×. Western Blotting Cells treated with CREM siRNA or control siRNA were lysed in a radioimmunoprecipitation assay buffer supplemented with protease inhibitors. Samples were normalized for protein concentration, and equal amounts of material in the Laemmli sample buffer were subjected to SDS-PAGE and transferred to nitrocellulose filters. For immunoblotting, the mouse monoclonal anti-CREM antibody (clone 3B; Novusbio, Littleton, CO) and the rabbit monoclonal anti-EWSR1 antibody (ab133288; Abcam, Cambridge, UK) were incubated overnight in a cold room at 1∶1000 dilution in 5% BSA/TBS/Tween 0.1%. After that, the membrane was incubated for 1 hr at room temperature with a mix of goat anti-rabbit StarBright Blue 520 and goat anti-mouse StarBright Blue 700, and hFAB rhodamine anti-GAPDH (Bio-Rad, Hercules, CA, USA) antibodies at 1:1000 dilution. Equal protein loading was evaluated by the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was washed 3× with TBST between incubations. Bound proteins were detected by fluorescence using ChemiDoc Gel Imaging System (Bio-Rad). Human Tissue Samples To explore CREM protein expression in normal human tissues, we built a TMA of 39 different human tissues in collaboration with Auria Biobank. For this, formalin-fixed paraffin-embedded (FFPE) archival tissue samples from Auria Biobank were used. All procedures were followed in accordance with the principles of the Helsinki Declaration of 1975. The study was authorized by the Hospital District of Southwest Finland (decision T3/2019) and the Auria Biobank steering committee (decision AB19-2770). Four patient samples of each location/tissue type were included, apart from male and female reproductive tissues, in which two samples each were included. The tissue samples had originally been taken surgically for diagnostic histological analysis at the Turku University Hospital Department of Pathology, except for the cerebrum, cerebellum, and heart samples, which were taken at autopsy. To identify potential samples, Auria Biobank sample and data archives were probed with SQL searches using SNOMED codes. Criteria for the samples were patients aged 16–75 years at the time of sampling and a desired topography. Reproductive tissues were from patients of the following age (years): fallopian tube fimbriae, 41–49; myometrium, 35–42; ovary, 30–34; secretory phase endometrium, 35–41; proliferative phase endometrium, 40–42; breast, 37–41; testis 49–62; and prostate, 70–73. H.K. and M.G. selected cases with essentially normal histology and made 1.5 mm diameter annotations to hematoxylin and eosin–stained scanned slides using CaseViewer software (3DHistech; Budapest, Hungary). Corresponding FFPE tissue cores were transferred into TMA blocks using TMA Grand Master (3DHistech). Bone marrow and breast tissue were used as whole sections due to technical reasons (adipose tissue and bone section poorly from a TMA block). Skin samples were used as whole sections to find a sufficient number of melanocytes for analysis. Bone marrow samples had been decalcified before paraffin embedding. Transcriptomic Analyses Publicly available databases were used to examine the expression of CREM mRNA. Normal tissue expression was investigated using the GTEx Portal V8 Release (GTEX database). For comparison of expression in normal tissue and cancer, we used the Gene Expression database of Normal and Tumor tissues 2 (GENT2; available at http://gent2.appex.kr/gent2/ ). Student’s t -test analysis was conducted using SPSS version 26 (IBM Corp.; Armonk, NY). Immunohistochemistry Paraffin blocks were sectioned at 4 µm. Immunostaining was performed using a LabVision autostainer (Thermo-Fisher Scientific, Waltham, MA). For antigen retrieval, Tris-EDTA pH 9.0 and microwave heating were used. Endogenous enzymes were blocked with hydrogen peroxidase, and a pre-protein block was done using Draco antibody diluent (WellMed AD125). The TMA sections were incubated for 60 min at room temperature with the mouse monoclonal anti-CREM antibody (clone 3B; Novusbio; 1:000 dilution) in Draco antibody diluent. The sections were then washed with 0.05 M Tris-HCl and treated with Orion II step detection system goat anti-mouse/rabbit secondary antibody [horseradish peroxidase (HRP), WellMed T100 HRP] at room temperature for 30 min. Counterstaining was done with Mayer’s hematoxylin, and finally, the sections were mounted with PERTEX. Evaluation of IHC The CREM-stained TMA slides were scanned using a Panoramic 250 Flash scanner (3DHistech) and histologically evaluated CaseViewer software (3DHistech). H.K. took pictures of each tissue sample from a representative area containing at least 100 cells of desired type (or if needed, more pictures were taken and the results were summed) and used ImmunoRatio software to calculate positive nuclear staining. Staining intensity was evaluated by H.K. and M.G. independently, and discrepancies were discussed and evaluated together. Intensity was scored using a four-tier grading system: 0 (negative), 1 (mild), 2 (moderate), and 3 (strong). The dominant staining intensity was evaluated and the percentage of positive nuclei was calculated for each sample separately. 

The commercially available human skin melanoma CHL-1, the human embryonic kidney HEK-293, and the human prostate carcinoma PC-3 were obtained from American Type Culture Collection (Manassas, VA). CHL-1 and HEK-293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 5 mM ultraglutamine, and 100 U/ml penicillin-streptomycin (Gibco; Carlsbad, CA), whereas PC-3 cells were cultured in RPMI medium with the same supplements. 

The CREM transcripts were knocked down in CHL-1, HEK-293, and PC-3 cells using 50 nM of human CREM small interfering RNA (siRNA) (sc-37700; Santa Cruz Biotechnology, Santa Cruz, CA) containing a pool of three to five target-specific 19–25 nucleotide sequences in length. A mix of three different non-targeting siRNAs (sc-37007, sc-44230, and sc-44231; Santa Cruz Biotechnology) was used as control. Cells were transfected using Dharmafect 4 transfection reagent (Dharmacon Research) according to the manufacturer’s instructions. The knockdown efficacy was examined 48 hr after transfection by Western blotting. The experiments were repeated at least 3×. 

Cells treated with CREM siRNA or control siRNA were lysed in a radioimmunoprecipitation assay buffer supplemented with protease inhibitors. Samples were normalized for protein concentration, and equal amounts of material in the Laemmli sample buffer were subjected to SDS-PAGE and transferred to nitrocellulose filters. For immunoblotting, the mouse monoclonal anti-CREM antibody (clone 3B; Novusbio, Littleton, CO) and the rabbit monoclonal anti-EWSR1 antibody (ab133288; Abcam, Cambridge, UK) were incubated overnight in a cold room at 1∶1000 dilution in 5% BSA/TBS/Tween 0.1%. After that, the membrane was incubated for 1 hr at room temperature with a mix of goat anti-rabbit StarBright Blue 520 and goat anti-mouse StarBright Blue 700, and hFAB rhodamine anti-GAPDH (Bio-Rad, Hercules, CA, USA) antibodies at 1:1000 dilution. Equal protein loading was evaluated by the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was washed 3× with TBST between incubations. Bound proteins were detected by fluorescence using ChemiDoc Gel Imaging System (Bio-Rad). 

To explore CREM protein expression in normal human tissues, we built a TMA of 39 different human tissues in collaboration with Auria Biobank. For this, formalin-fixed paraffin-embedded (FFPE) archival tissue samples from Auria Biobank were used. All procedures were followed in accordance with the principles of the Helsinki Declaration of 1975. The study was authorized by the Hospital District of Southwest Finland (decision T3/2019) and the Auria Biobank steering committee (decision AB19-2770). Four patient samples of each location/tissue type were included, apart from male and female reproductive tissues, in which two samples each were included. The tissue samples had originally been taken surgically for diagnostic histological analysis at the Turku University Hospital Department of Pathology, except for the cerebrum, cerebellum, and heart samples, which were taken at autopsy. To identify potential samples, Auria Biobank sample and data archives were probed with SQL searches using SNOMED codes. Criteria for the samples were patients aged 16–75 years at the time of sampling and a desired topography. Reproductive tissues were from patients of the following age (years): fallopian tube fimbriae, 41–49; myometrium, 35–42; ovary, 30–34; secretory phase endometrium, 35–41; proliferative phase endometrium, 40–42; breast, 37–41; testis 49–62; and prostate, 70–73. H.K. and M.G. selected cases with essentially normal histology and made 1.5 mm diameter annotations to hematoxylin and eosin–stained scanned slides using CaseViewer software (3DHistech; Budapest, Hungary). Corresponding FFPE tissue cores were transferred into TMA blocks using TMA Grand Master (3DHistech). Bone marrow and breast tissue were used as whole sections due to technical reasons (adipose tissue and bone section poorly from a TMA block). Skin samples were used as whole sections to find a sufficient number of melanocytes for analysis. Bone marrow samples had been decalcified before paraffin embedding. 

Publicly available databases were used to examine the expression of CREM mRNA. Normal tissue expression was investigated using the GTEx Portal V8 Release (GTEX database). For comparison of expression in normal tissue and cancer, we used the Gene Expression database of Normal and Tumor tissues 2 (GENT2; available at http://gent2.appex.kr/gent2/ ). Student’s t -test analysis was conducted using SPSS version 26 (IBM Corp.; Armonk, NY). 

Paraffin blocks were sectioned at 4 µm. Immunostaining was performed using a LabVision autostainer (Thermo-Fisher Scientific, Waltham, MA). For antigen retrieval, Tris-EDTA pH 9.0 and microwave heating were used. Endogenous enzymes were blocked with hydrogen peroxidase, and a pre-protein block was done using Draco antibody diluent (WellMed AD125). The TMA sections were incubated for 60 min at room temperature with the mouse monoclonal anti-CREM antibody (clone 3B; Novusbio; 1:000 dilution) in Draco antibody diluent. The sections were then washed with 0.05 M Tris-HCl and treated with Orion II step detection system goat anti-mouse/rabbit secondary antibody [horseradish peroxidase (HRP), WellMed T100 HRP] at room temperature for 30 min. Counterstaining was done with Mayer’s hematoxylin, and finally, the sections were mounted with PERTEX. 

The CREM-stained TMA slides were scanned using a Panoramic 250 Flash scanner (3DHistech) and histologically evaluated CaseViewer software (3DHistech). H.K. took pictures of each tissue sample from a representative area containing at least 100 cells of desired type (or if needed, more pictures were taken and the results were summed) and used ImmunoRatio software to calculate positive nuclear staining. Staining intensity was evaluated by H.K. and M.G. independently, and discrepancies were discussed and evaluated together. Intensity was scored using a four-tier grading system: 0 (negative), 1 (mild), 2 (moderate), and 3 (strong). The dominant staining intensity was evaluated and the percentage of positive nuclei was calculated for each sample separately. 

Validation of Antibody First, we wanted to ensure that the antibody we used specifically detects the CREM protein. This commercially available mouse monoclonal anti-human CREM antibody (clone 3B; Novusbio) had been generated using a target sequence containing amino acids 201–300 of the protein, which includes the DNA-binding domain of the CREM protein. A schematic presentation of the CREM gene, protein, and the antibody target sequence is presented in . This antibody could detect 28 of the 29 CREM isoforms with sequence identity of 85–100%. The sequence homology with members of the same transcription family CREB and ATF is low (67% and 57%, respectively). First, we performed Western blotting using CREM antibody on cell lysates from human skin melanoma CHL-1, human embryonic kidney HEK-293, and prostate cancer PC-3 cell lines. The cells had been treated with CREM-targeting siRNA or control siRNA. Two or three major bands of expected size (≈37, 30, and 20 kDa) were detected in the immunoblots of all control cell line lysates ( ). Furthermore, we wanted to test whether the CREM antibody would detect an EWSR1-CREM fusion in the CHL-1 cell line, which is known to harbor this rearrangement. Western blotting using the anti-CREM or anti-EWSR1 antibody detected a band (≈55 kDa) not corresponding to the predicted size of CREM. The two secondary antibodies were then labeled with different fluorescence labels, and in double immunofluorescent blotting they co-localized in the same band identifying it as the EWSR1-CREM fusion protein. The fusion protein band was markedly weaker after CREM siRNA treatment, indicating successful knockdown. Also the bands corresponding to wild-type CREM protein were weaker in all three cell lines after CREM knockdown. Endogenous EWSR1 (which is ubiquitously expressed) was not affected by CREM knockdown. Taken together, these findings agree that the CREM antibody specifically detects CREM protein. Analysis of CREM mRNA Expression in Human Tissue From Databases To get an overview of CREM mRNA expression that we could later compare protein expression with, we studied the expression of CREM mRNA in different human tissues using the GTEx database. The expression was calculated from a gene model with isoforms collapsed to a single gene (the GTEX database can also be used to investigate isoform-specific expression). In this analysis, the highest expression of CREM was seen in the testis, followed by the adrenal gland, the placenta, and the appendix. Most tissues expressed CREM at moderate or low levels. The least expression was seen in the pancreas, followed by the skeletal muscle, the salivary gland, and the skin ( ). Immunohistochemical Analysis of CREM in Human Tissues To gain detailed knowledge of which specific cell types in various organ systems express the CREM protein, an immunohistochemical analysis was performed on TMAs with several samples of 39 normal human tissues. Staining intensity was divided into four groups: negative, weak, moderate, and strong. The CREM protein expression is presented as organ system groups below, and the results are summarized in . The staining intensity evaluation scheme is presented in , and representative images of the selected tissues are presented in to . As a rule, CREM expression was wide, as it was found in almost all tissues. As expected of a transcription factor, CREM was primarily localized in the nuclei of almost all cell types. In most tissues, expression was extensive with positivity in parenchymal cells and supporting structures such as stromal and endothelial cells. Mitotic cells were negative, when present. Mature red blood cells were present in many samples, and they did not express CREM. Most of the tissues had some cytoplasmic staining of different intensities ( ). Central Nervous System In the cerebrum and cerebellum, both cortical neurons and glial cells had virtually no expression of CREM ( ). The molecular layer of the cerebellum showed moderate fibrillar staining. Some mild to moderate staining was noted in the endothelial cells of capillaries of the cerebral white matter. Respiratory System In the nasal cavity, CREM expression in the respiratory epithelium was extensive and of moderate intensity ( ). The stroma had moderate to high staining intensity. Below the surface epithelium, both mucinous and serous glands were stained with a moderate intensity. However, the number of cells expressing CREM was dissimilar: in mucinous glands 19–21% and in serous glands 78–87%. The bronchial epithelium had CREM expression of mild to moderate staining intensity. The alveolar epithelium of the lung had extensive CREM expression, in 75–80% of pneumocytes. No significant difference between pneumocytes type I and II was detected. Gastrointestinal Tract and Organs The stratified non-keratinizing squamous cells of the palate expressed CREM moderately, with positive staining in 60–67% of cells. The nuclei lost their CREM expression as they matured so that usually the upper third or upper quarter of the epithelium was negative ( ). The squamous epithelium of the floor of the mouth had a very similar pattern, with slightly weaker expression overall. The same gradient pattern was detected also in the stratified squamous epithelium of the tongue, and the CREM expression varied, with 21–68% of cells being positive with a dominantly low intensity. Two thirds of the esophageal squamous cell nuclei expressed CREM with a low to moderate intensity. In this epithelium, the positive nuclei were evenly distributed within the epithelium. The stroma had strong expression of CREM, with positivity in 53–73%. In gastric mucosa, 53–75% of cells expressed CREM. The surface epithelium of the gastric corpus expressed CREM with the exception of apical mucous cells, which were negative. The glandular cells of the gastric corpus showed expression of CREM with weak to moderate intensity ( ). The duodenal epithelium had a mild staining intensity with various range of CREM expression. Enterocytes near the bottom of the crypts tended to be more often negative than apical enterocytes, and Paneth cells were mostly positive. In the stromal cells, we observed a weak to moderate staining intensity in about half of the cell nuclei. Half of the Brunner gland cell nuclei showed CREM expression, and it was of moderate intensity ( ). Further along the small intestine, 41–89% of the epithelium expressed CREM with a weak intensity. Goblet cells were negative, and the endocrine cells stained similarly as the enterocytes. In all, 62–83% of the stromal cells had CREM expression of weak to moderate intensity. As in the small intestine, in the colon two thirds of the mucosa had expression of CREM with a weak intensity. Here, the enterocytes in the crypts tended to be more negative than apical cells and the endocrine cells stained similarly as the cryptal enterocytes. Goblet cells were more often negative than the enterocytes. The nuclei in the stroma had moderate to strong staining intensity and a wide expression of CREM. The acinar and ductal cells in the parotid gland had CREM expression of low to moderate intensity. In the submandibular gland, approximately half of the mucous gland cells had weak staining intensity. On the contrary, nuclei of the serous glands had moderate to strong staining intensity of CREM. Most of the ductal cells showed intensive staining ( ). In the pancreas, both exocrine and the islets of Langerhans endocrine cell nuclei were widely positive with weak intensity (68–83% and 61–72%, respectively). Pancreatic tissue is depicted in , as an example of weak staining intensity. In the liver, the hepatocyte nuclei were uniformly negative. Their cytoplasm had some moderate granular staining ( ). The biliary duct cells were negative apart from one sample with weak uniform expression of CREM. The gallbladder epithelium consisting of columnar cells had weak to moderate staining in most of the nuclei. Of the stromal cells, less than half of the nuclei had moderate to strong staining intensity. Endocrine Organs Most of the thyroid follicular cells had moderate to strong staining intensity. In the parathyroid gland, the chief cells and oxyphil cells had a moderate to strong staining intensity in 52–88% of cells. The parathyroid tissue is depicted in , as an example of strong staining intensity. Adrenal gland cortical cells had a strong and a rather uniform staining pattern. Urinary System The range of positively staining glomerular cells in the kidney varied from 40% to 60%, and the staining intensity was weak to moderate. The tubular cells were nearly always positive so that the proximal tubules had weak and the distal tubules had moderate staining ( ). In the urinary bladder, 79–91% of the urothelial cells had a weak to moderate staining intensity. The stromal cells had a moderate staining CREM staining intensity in 50–83% of nuclei ( ). Female Reproductive System In the non-lactating female breast, the ductal and lobular epithelium stained alike. They had weak to moderate intensity and a near 80% range of CREM expression. The luminal cell nuclei had slightly less intense staining compared with the basal cell nuclei. In all, 90% of the glands of the proliferative endometrium had weak immunoreactivity. The stroma also showed CREM expression, with weak to moderate staining intensity in 86%. The capillary endothelial cells had a moderate to strong staining intensity and were easily distinguishable in the stroma. A varied expression of CREM was detected in the secretory endometrium. Glandular cells and stroma had weak to moderate staining intensity in 70% and 64% of nuclei, respectively. The smooth muscle cells of the myometrium had a weak to moderate immunoreactivity in 74–78%. The ciliated columnar epithelium of the fallopian tube fimbriae had a strong and uniform staining of the nuclei ( ). In the ovaries, the stromal cells stained variably, ranging from negative to moderate intensity. Primordial follicles had uniform moderate CREM expression and the granulosa cells stained partially with moderate intensity. The late third trimester (H38–39) placenta had negative syncytiotrophoblasts. Half of the cytotrophoblasts had moderate-intensity CREM expression in the villi ( ). Male Reproductive System The prostate gland epithelium had weak CREM expression in 58–93% of nuclei. The luminal secretory cells stained with similar intensity. In the seminiferous tubules of the testis, 74–96% of cells were positive with strong intensity. The spermatids had no CREM expression, whereas the Sertoli cells and spermatocytes had staining intensity from negative to strong. Leydig cells had a weak staining intensity ( ). Hematopoietic and Lymphatic System For bone marrow to be sectioned for histological analysis, it is routinely decalcified to make it softer for sectioning. In this way, the bone marrow had an additional step in tissue preparation compared with the other tissues in this study. Bone marrow cell types were identified based on their morphology. Cells of the bone marrow had overall a sparse CREM expression. The erythroid cells were all negative, and the cells of granulopoietic lineage were mainly negative and the megakaryocytes mainly positive with mild staining intensity. Red pulp of the spleen had moderate (to strong) staining intensity of the sinusoidal cell nuclei, whereas the chordal macrophages/pulp chords remained negative. The white pulp had sprinkles of CREM expression of varying intensity ( ). The germinal centers of the lymph node cortex had moderate and fairly uniform expression of CREM in contrast to the surrounding cortical mature lymphocytes of which only half had CREM expression of mild to moderate intensity ( ). In the tonsil, we observed a similar pattern as in the lymph node; the germinal centers had a much wider and stronger CREM expression than the mature lymphocytes. The non-keratinizing stratified squamous cell epithelium had a positive basal layer with nuclei losing the CREM expression as they matured toward the surface. Skin The keratinizing stratified squamous cell nuclei in the skin epidermis had mild to moderate immunoreactivity in little over half of the cells. In a vast majority of melanocytes, no CREM expression could be detected. Weak staining was seen in 0–11% of nuclei. Eccrine glands and sebaceous glands showed moderate staining intensity in nearly all of the cell nuclei. Cardiac and Striated Muscle In myocytes of the heart, we detected no CREM expression. Striated myocytes of the skeletal muscles had extensive expression of CREM with weak to moderate intensity ( ). CREM Expression in Cancer Next, we studied whether CREM expression is altered in cancer. For this, we used the GENT2 database (publicly available at http://gent2.appex.kr/gent2/ ). In this analysis, we found CREM mRNA expression in all forms of cancer, often with slightly different expression levels compared with normal tissues ( ). A slight upregulation was seen in cancers of the immune system and skin cancer. Conversely, in most forms of cancer, downregulation of CREM expression was noted. This was most apparent in cancers of the testis, the urinary bladder, and the breast. Furthermore, there was a large number of outliers in several forms of cancer, with substantially higher or lower expression than in the majority of cases. The difference between CREM mRNA level in normal tissues and cancer was statistically significant in all organs except colon, skin, and stomach. Second, we wanted to examine whether a tumor with an EWSR1-CREM fusion gene might overexpress the fusion protein, resulting in stronger immunohistochemical CREM staining intensity than seen in the corresponding normal tissues. To test this, five low-grade MECs were immunohistochemically stained and analyzed. One of these had an EWSR-CREM fusion gene, detected by RNA next-generation sequencing and confirmed by RT-PCR using the methodology described previously. The EWSR1 rearrangement had been confirmed by fluorescence in situ hybridization analysis, using an Abbott Vysis EWSR1 break-apart probe. In the immunohistochemical analysis, we found moderate positive CREM staining homogeneously in most tumor cell nuclei in all five cases (80–88% of cells). This pattern was slightly different from most normal tissues, in which variation in staining intensity as well as negative nuclei were more often present. The MEC with an EWSR1-CREM fusion gene could not by CREM staining be distinguished from ones that did not harbor this fusion gene ( and ). These findings show that CREM protein is moderately expressed in MEC, and that CREM IHC cannot be used as a surrogate marker in detecting CREM fusion genes in this context. 

First, we wanted to ensure that the antibody we used specifically detects the CREM protein. This commercially available mouse monoclonal anti-human CREM antibody (clone 3B; Novusbio) had been generated using a target sequence containing amino acids 201–300 of the protein, which includes the DNA-binding domain of the CREM protein. A schematic presentation of the CREM gene, protein, and the antibody target sequence is presented in . This antibody could detect 28 of the 29 CREM isoforms with sequence identity of 85–100%. The sequence homology with members of the same transcription family CREB and ATF is low (67% and 57%, respectively). First, we performed Western blotting using CREM antibody on cell lysates from human skin melanoma CHL-1, human embryonic kidney HEK-293, and prostate cancer PC-3 cell lines. The cells had been treated with CREM-targeting siRNA or control siRNA. Two or three major bands of expected size (≈37, 30, and 20 kDa) were detected in the immunoblots of all control cell line lysates ( ). Furthermore, we wanted to test whether the CREM antibody would detect an EWSR1-CREM fusion in the CHL-1 cell line, which is known to harbor this rearrangement. Western blotting using the anti-CREM or anti-EWSR1 antibody detected a band (≈55 kDa) not corresponding to the predicted size of CREM. The two secondary antibodies were then labeled with different fluorescence labels, and in double immunofluorescent blotting they co-localized in the same band identifying it as the EWSR1-CREM fusion protein. The fusion protein band was markedly weaker after CREM siRNA treatment, indicating successful knockdown. Also the bands corresponding to wild-type CREM protein were weaker in all three cell lines after CREM knockdown. Endogenous EWSR1 (which is ubiquitously expressed) was not affected by CREM knockdown. Taken together, these findings agree that the CREM antibody specifically detects CREM protein. 

To get an overview of CREM mRNA expression that we could later compare protein expression with, we studied the expression of CREM mRNA in different human tissues using the GTEx database. The expression was calculated from a gene model with isoforms collapsed to a single gene (the GTEX database can also be used to investigate isoform-specific expression). In this analysis, the highest expression of CREM was seen in the testis, followed by the adrenal gland, the placenta, and the appendix. Most tissues expressed CREM at moderate or low levels. The least expression was seen in the pancreas, followed by the skeletal muscle, the salivary gland, and the skin ( ). 

To gain detailed knowledge of which specific cell types in various organ systems express the CREM protein, an immunohistochemical analysis was performed on TMAs with several samples of 39 normal human tissues. Staining intensity was divided into four groups: negative, weak, moderate, and strong. The CREM protein expression is presented as organ system groups below, and the results are summarized in . The staining intensity evaluation scheme is presented in , and representative images of the selected tissues are presented in to . As a rule, CREM expression was wide, as it was found in almost all tissues. As expected of a transcription factor, CREM was primarily localized in the nuclei of almost all cell types. In most tissues, expression was extensive with positivity in parenchymal cells and supporting structures such as stromal and endothelial cells. Mitotic cells were negative, when present. Mature red blood cells were present in many samples, and they did not express CREM. Most of the tissues had some cytoplasmic staining of different intensities ( ). Central Nervous System In the cerebrum and cerebellum, both cortical neurons and glial cells had virtually no expression of CREM ( ). The molecular layer of the cerebellum showed moderate fibrillar staining. Some mild to moderate staining was noted in the endothelial cells of capillaries of the cerebral white matter. Respiratory System In the nasal cavity, CREM expression in the respiratory epithelium was extensive and of moderate intensity ( ). The stroma had moderate to high staining intensity. Below the surface epithelium, both mucinous and serous glands were stained with a moderate intensity. However, the number of cells expressing CREM was dissimilar: in mucinous glands 19–21% and in serous glands 78–87%. The bronchial epithelium had CREM expression of mild to moderate staining intensity. The alveolar epithelium of the lung had extensive CREM expression, in 75–80% of pneumocytes. No significant difference between pneumocytes type I and II was detected. Gastrointestinal Tract and Organs The stratified non-keratinizing squamous cells of the palate expressed CREM moderately, with positive staining in 60–67% of cells. The nuclei lost their CREM expression as they matured so that usually the upper third or upper quarter of the epithelium was negative ( ). The squamous epithelium of the floor of the mouth had a very similar pattern, with slightly weaker expression overall. The same gradient pattern was detected also in the stratified squamous epithelium of the tongue, and the CREM expression varied, with 21–68% of cells being positive with a dominantly low intensity. Two thirds of the esophageal squamous cell nuclei expressed CREM with a low to moderate intensity. In this epithelium, the positive nuclei were evenly distributed within the epithelium. The stroma had strong expression of CREM, with positivity in 53–73%. In gastric mucosa, 53–75% of cells expressed CREM. The surface epithelium of the gastric corpus expressed CREM with the exception of apical mucous cells, which were negative. The glandular cells of the gastric corpus showed expression of CREM with weak to moderate intensity ( ). The duodenal epithelium had a mild staining intensity with various range of CREM expression. Enterocytes near the bottom of the crypts tended to be more often negative than apical enterocytes, and Paneth cells were mostly positive. In the stromal cells, we observed a weak to moderate staining intensity in about half of the cell nuclei. Half of the Brunner gland cell nuclei showed CREM expression, and it was of moderate intensity ( ). Further along the small intestine, 41–89% of the epithelium expressed CREM with a weak intensity. Goblet cells were negative, and the endocrine cells stained similarly as the enterocytes. In all, 62–83% of the stromal cells had CREM expression of weak to moderate intensity. As in the small intestine, in the colon two thirds of the mucosa had expression of CREM with a weak intensity. Here, the enterocytes in the crypts tended to be more negative than apical cells and the endocrine cells stained similarly as the cryptal enterocytes. Goblet cells were more often negative than the enterocytes. The nuclei in the stroma had moderate to strong staining intensity and a wide expression of CREM. The acinar and ductal cells in the parotid gland had CREM expression of low to moderate intensity. In the submandibular gland, approximately half of the mucous gland cells had weak staining intensity. On the contrary, nuclei of the serous glands had moderate to strong staining intensity of CREM. Most of the ductal cells showed intensive staining ( ). In the pancreas, both exocrine and the islets of Langerhans endocrine cell nuclei were widely positive with weak intensity (68–83% and 61–72%, respectively). Pancreatic tissue is depicted in , as an example of weak staining intensity. In the liver, the hepatocyte nuclei were uniformly negative. Their cytoplasm had some moderate granular staining ( ). The biliary duct cells were negative apart from one sample with weak uniform expression of CREM. The gallbladder epithelium consisting of columnar cells had weak to moderate staining in most of the nuclei. Of the stromal cells, less than half of the nuclei had moderate to strong staining intensity. Endocrine Organs Most of the thyroid follicular cells had moderate to strong staining intensity. In the parathyroid gland, the chief cells and oxyphil cells had a moderate to strong staining intensity in 52–88% of cells. The parathyroid tissue is depicted in , as an example of strong staining intensity. Adrenal gland cortical cells had a strong and a rather uniform staining pattern. Urinary System The range of positively staining glomerular cells in the kidney varied from 40% to 60%, and the staining intensity was weak to moderate. The tubular cells were nearly always positive so that the proximal tubules had weak and the distal tubules had moderate staining ( ). In the urinary bladder, 79–91% of the urothelial cells had a weak to moderate staining intensity. The stromal cells had a moderate staining CREM staining intensity in 50–83% of nuclei ( ). Female Reproductive System In the non-lactating female breast, the ductal and lobular epithelium stained alike. They had weak to moderate intensity and a near 80% range of CREM expression. The luminal cell nuclei had slightly less intense staining compared with the basal cell nuclei. In all, 90% of the glands of the proliferative endometrium had weak immunoreactivity. The stroma also showed CREM expression, with weak to moderate staining intensity in 86%. The capillary endothelial cells had a moderate to strong staining intensity and were easily distinguishable in the stroma. A varied expression of CREM was detected in the secretory endometrium. Glandular cells and stroma had weak to moderate staining intensity in 70% and 64% of nuclei, respectively. The smooth muscle cells of the myometrium had a weak to moderate immunoreactivity in 74–78%. The ciliated columnar epithelium of the fallopian tube fimbriae had a strong and uniform staining of the nuclei ( ). In the ovaries, the stromal cells stained variably, ranging from negative to moderate intensity. Primordial follicles had uniform moderate CREM expression and the granulosa cells stained partially with moderate intensity. The late third trimester (H38–39) placenta had negative syncytiotrophoblasts. Half of the cytotrophoblasts had moderate-intensity CREM expression in the villi ( ). Male Reproductive System The prostate gland epithelium had weak CREM expression in 58–93% of nuclei. The luminal secretory cells stained with similar intensity. In the seminiferous tubules of the testis, 74–96% of cells were positive with strong intensity. The spermatids had no CREM expression, whereas the Sertoli cells and spermatocytes had staining intensity from negative to strong. Leydig cells had a weak staining intensity ( ). Hematopoietic and Lymphatic System For bone marrow to be sectioned for histological analysis, it is routinely decalcified to make it softer for sectioning. In this way, the bone marrow had an additional step in tissue preparation compared with the other tissues in this study. Bone marrow cell types were identified based on their morphology. Cells of the bone marrow had overall a sparse CREM expression. The erythroid cells were all negative, and the cells of granulopoietic lineage were mainly negative and the megakaryocytes mainly positive with mild staining intensity. Red pulp of the spleen had moderate (to strong) staining intensity of the sinusoidal cell nuclei, whereas the chordal macrophages/pulp chords remained negative. The white pulp had sprinkles of CREM expression of varying intensity ( ). The germinal centers of the lymph node cortex had moderate and fairly uniform expression of CREM in contrast to the surrounding cortical mature lymphocytes of which only half had CREM expression of mild to moderate intensity ( ). In the tonsil, we observed a similar pattern as in the lymph node; the germinal centers had a much wider and stronger CREM expression than the mature lymphocytes. The non-keratinizing stratified squamous cell epithelium had a positive basal layer with nuclei losing the CREM expression as they matured toward the surface. Skin The keratinizing stratified squamous cell nuclei in the skin epidermis had mild to moderate immunoreactivity in little over half of the cells. In a vast majority of melanocytes, no CREM expression could be detected. Weak staining was seen in 0–11% of nuclei. Eccrine glands and sebaceous glands showed moderate staining intensity in nearly all of the cell nuclei. Cardiac and Striated Muscle In myocytes of the heart, we detected no CREM expression. Striated myocytes of the skeletal muscles had extensive expression of CREM with weak to moderate intensity ( ). 

In the cerebrum and cerebellum, both cortical neurons and glial cells had virtually no expression of CREM ( ). The molecular layer of the cerebellum showed moderate fibrillar staining. Some mild to moderate staining was noted in the endothelial cells of capillaries of the cerebral white matter. 

In the nasal cavity, CREM expression in the respiratory epithelium was extensive and of moderate intensity ( ). The stroma had moderate to high staining intensity. Below the surface epithelium, both mucinous and serous glands were stained with a moderate intensity. However, the number of cells expressing CREM was dissimilar: in mucinous glands 19–21% and in serous glands 78–87%. The bronchial epithelium had CREM expression of mild to moderate staining intensity. The alveolar epithelium of the lung had extensive CREM expression, in 75–80% of pneumocytes. No significant difference between pneumocytes type I and II was detected. 

The stratified non-keratinizing squamous cells of the palate expressed CREM moderately, with positive staining in 60–67% of cells. The nuclei lost their CREM expression as they matured so that usually the upper third or upper quarter of the epithelium was negative ( ). The squamous epithelium of the floor of the mouth had a very similar pattern, with slightly weaker expression overall. The same gradient pattern was detected also in the stratified squamous epithelium of the tongue, and the CREM expression varied, with 21–68% of cells being positive with a dominantly low intensity. Two thirds of the esophageal squamous cell nuclei expressed CREM with a low to moderate intensity. In this epithelium, the positive nuclei were evenly distributed within the epithelium. The stroma had strong expression of CREM, with positivity in 53–73%. In gastric mucosa, 53–75% of cells expressed CREM. The surface epithelium of the gastric corpus expressed CREM with the exception of apical mucous cells, which were negative. The glandular cells of the gastric corpus showed expression of CREM with weak to moderate intensity ( ). The duodenal epithelium had a mild staining intensity with various range of CREM expression. Enterocytes near the bottom of the crypts tended to be more often negative than apical enterocytes, and Paneth cells were mostly positive. In the stromal cells, we observed a weak to moderate staining intensity in about half of the cell nuclei. Half of the Brunner gland cell nuclei showed CREM expression, and it was of moderate intensity ( ). Further along the small intestine, 41–89% of the epithelium expressed CREM with a weak intensity. Goblet cells were negative, and the endocrine cells stained similarly as the enterocytes. In all, 62–83% of the stromal cells had CREM expression of weak to moderate intensity. As in the small intestine, in the colon two thirds of the mucosa had expression of CREM with a weak intensity. Here, the enterocytes in the crypts tended to be more negative than apical cells and the endocrine cells stained similarly as the cryptal enterocytes. Goblet cells were more often negative than the enterocytes. The nuclei in the stroma had moderate to strong staining intensity and a wide expression of CREM. The acinar and ductal cells in the parotid gland had CREM expression of low to moderate intensity. In the submandibular gland, approximately half of the mucous gland cells had weak staining intensity. On the contrary, nuclei of the serous glands had moderate to strong staining intensity of CREM. Most of the ductal cells showed intensive staining ( ). In the pancreas, both exocrine and the islets of Langerhans endocrine cell nuclei were widely positive with weak intensity (68–83% and 61–72%, respectively). Pancreatic tissue is depicted in , as an example of weak staining intensity. In the liver, the hepatocyte nuclei were uniformly negative. Their cytoplasm had some moderate granular staining ( ). The biliary duct cells were negative apart from one sample with weak uniform expression of CREM. The gallbladder epithelium consisting of columnar cells had weak to moderate staining in most of the nuclei. Of the stromal cells, less than half of the nuclei had moderate to strong staining intensity. 

Most of the thyroid follicular cells had moderate to strong staining intensity. In the parathyroid gland, the chief cells and oxyphil cells had a moderate to strong staining intensity in 52–88% of cells. The parathyroid tissue is depicted in , as an example of strong staining intensity. Adrenal gland cortical cells had a strong and a rather uniform staining pattern. 

The range of positively staining glomerular cells in the kidney varied from 40% to 60%, and the staining intensity was weak to moderate. The tubular cells were nearly always positive so that the proximal tubules had weak and the distal tubules had moderate staining ( ). In the urinary bladder, 79–91% of the urothelial cells had a weak to moderate staining intensity. The stromal cells had a moderate staining CREM staining intensity in 50–83% of nuclei ( ). 

In the non-lactating female breast, the ductal and lobular epithelium stained alike. They had weak to moderate intensity and a near 80% range of CREM expression. The luminal cell nuclei had slightly less intense staining compared with the basal cell nuclei. In all, 90% of the glands of the proliferative endometrium had weak immunoreactivity. The stroma also showed CREM expression, with weak to moderate staining intensity in 86%. The capillary endothelial cells had a moderate to strong staining intensity and were easily distinguishable in the stroma. A varied expression of CREM was detected in the secretory endometrium. Glandular cells and stroma had weak to moderate staining intensity in 70% and 64% of nuclei, respectively. The smooth muscle cells of the myometrium had a weak to moderate immunoreactivity in 74–78%. The ciliated columnar epithelium of the fallopian tube fimbriae had a strong and uniform staining of the nuclei ( ). In the ovaries, the stromal cells stained variably, ranging from negative to moderate intensity. Primordial follicles had uniform moderate CREM expression and the granulosa cells stained partially with moderate intensity. The late third trimester (H38–39) placenta had negative syncytiotrophoblasts. Half of the cytotrophoblasts had moderate-intensity CREM expression in the villi ( ). 

The prostate gland epithelium had weak CREM expression in 58–93% of nuclei. The luminal secretory cells stained with similar intensity. In the seminiferous tubules of the testis, 74–96% of cells were positive with strong intensity. The spermatids had no CREM expression, whereas the Sertoli cells and spermatocytes had staining intensity from negative to strong. Leydig cells had a weak staining intensity ( ). 

For bone marrow to be sectioned for histological analysis, it is routinely decalcified to make it softer for sectioning. In this way, the bone marrow had an additional step in tissue preparation compared with the other tissues in this study. Bone marrow cell types were identified based on their morphology. Cells of the bone marrow had overall a sparse CREM expression. The erythroid cells were all negative, and the cells of granulopoietic lineage were mainly negative and the megakaryocytes mainly positive with mild staining intensity. Red pulp of the spleen had moderate (to strong) staining intensity of the sinusoidal cell nuclei, whereas the chordal macrophages/pulp chords remained negative. The white pulp had sprinkles of CREM expression of varying intensity ( ). The germinal centers of the lymph node cortex had moderate and fairly uniform expression of CREM in contrast to the surrounding cortical mature lymphocytes of which only half had CREM expression of mild to moderate intensity ( ). In the tonsil, we observed a similar pattern as in the lymph node; the germinal centers had a much wider and stronger CREM expression than the mature lymphocytes. The non-keratinizing stratified squamous cell epithelium had a positive basal layer with nuclei losing the CREM expression as they matured toward the surface. 

The keratinizing stratified squamous cell nuclei in the skin epidermis had mild to moderate immunoreactivity in little over half of the cells. In a vast majority of melanocytes, no CREM expression could be detected. Weak staining was seen in 0–11% of nuclei. Eccrine glands and sebaceous glands showed moderate staining intensity in nearly all of the cell nuclei. 

In myocytes of the heart, we detected no CREM expression. Striated myocytes of the skeletal muscles had extensive expression of CREM with weak to moderate intensity ( ). 

Next, we studied whether CREM expression is altered in cancer. For this, we used the GENT2 database (publicly available at http://gent2.appex.kr/gent2/ ). In this analysis, we found CREM mRNA expression in all forms of cancer, often with slightly different expression levels compared with normal tissues ( ). A slight upregulation was seen in cancers of the immune system and skin cancer. Conversely, in most forms of cancer, downregulation of CREM expression was noted. This was most apparent in cancers of the testis, the urinary bladder, and the breast. Furthermore, there was a large number of outliers in several forms of cancer, with substantially higher or lower expression than in the majority of cases. The difference between CREM mRNA level in normal tissues and cancer was statistically significant in all organs except colon, skin, and stomach. Second, we wanted to examine whether a tumor with an EWSR1-CREM fusion gene might overexpress the fusion protein, resulting in stronger immunohistochemical CREM staining intensity than seen in the corresponding normal tissues. To test this, five low-grade MECs were immunohistochemically stained and analyzed. One of these had an EWSR-CREM fusion gene, detected by RNA next-generation sequencing and confirmed by RT-PCR using the methodology described previously. The EWSR1 rearrangement had been confirmed by fluorescence in situ hybridization analysis, using an Abbott Vysis EWSR1 break-apart probe. In the immunohistochemical analysis, we found moderate positive CREM staining homogeneously in most tumor cell nuclei in all five cases (80–88% of cells). This pattern was slightly different from most normal tissues, in which variation in staining intensity as well as negative nuclei were more often present. The MEC with an EWSR1-CREM fusion gene could not by CREM staining be distinguished from ones that did not harbor this fusion gene ( and ). These findings show that CREM protein is moderately expressed in MEC, and that CREM IHC cannot be used as a surrogate marker in detecting CREM fusion genes in this context. 

In this study, we analyzed the expression of CREM protein in 142 samples from 39 tissues. To our knowledge, this is the first systematic study addressing the expression of nearly all CREM isoforms, including the isoform commonly known as ICER. Previously, the Human Protein Atlas (available from http://www.proteinatlas.org ) has shown CREM stainings in human tissues using a polyclonal antibody that targets the N-terminal part of the canonical CREM protein expressed almost exclusively in the spermatogenic cells of the testis. The N-terminal domain of the canonical CREM protein is not present in recently discovered fusion gene products. Given that the antibody used in this study detects almost all CREM isoforms, a wider expression was not an unexpected finding. We found the expression of CREM in normal human tissues to be wide with only a few exceptions. The staining intensity and differences between cell types within tissues provided more variation and gave more detailed knowledge of CREM expression than mRNA-based tissue analyses. When mRNA levels are studied, a bulk extraction of the tissue is done; thus, it is a mix of all the cell types within the tissue. IHC is much more precise as it can truly show the protein expression of each cell and even the subcellular location. As an example, lymphatic tissues showed moderate expression of mRNA, whereas the protein expression had clear topographic differences. In the lymph node cortex and tonsils, we saw a very clear difference in CREM expression between germinal centers, where expression was strong, and the surrounding mature lymphocytes, which were weak or negative. The expression of CREM in immature B-lymphocytes and loss of expression in mature B-lymphocytes are suggestive of a role in B-cell proliferation and maturation. At large, the immunohistochemical results were in line with findings from mRNA expression databases. The strongest and most wide staining intensity was seen in reproductive tract tissues: the seminiferous tubules and the fallopian tube fimbrial epithelium. Similar strong staining was also present in salivary gland serous glands, parathyroid glands, and thyroid follicles. Most of the tissues expressed CREM moderately. In the least expressing end of the spectrum, the placental syncytiotrophoblast cells, and the mucus-secreting goblet cells in the epithelial tissues, CREM was virtually undetectable. The mucous glands had less CREM expression profiles than the other cell types of the same tissue. In the liver, hepatocytes had negative nuclei, but a fairly strong granular cytoplasmic staining which could explain the mRNA expression results that indicate positive expression. Tissues where the immunohistochemical results did not mirror transcription were the CNS and the heart, which had virtually no detectable expression by IHC. This discrepancy may in part be because these tissues were the sole autopsy samples in our study—the inevitable slight delay before fixation of autopsy tissues may have led to degradation of CREM. From other studies we know that CREM is expressed in the CNS, at least regionally. Miller et al conducted an intriguing study in mouse and human where they demonstrated that CREM is a mediator of impulsive action in the basal forebrain, specifically in the nucleus accumbens core (AcbC). The nucleus accumbens is known for its role in addiction. They further discovered an association between low CREM expression in the AcbC and a higher susceptibility to heroin abuse. Our cerebral samples were, in contrast, all cortical. In another study, the cerebral cortex CREM has been shown to be low, but it increased after neuronal damage. A wider set of tissue samples would be needed to thoroughly characterize CREM protein expression in the CNS. In several types of cancer, IHC can be used instead of genetic testing to detect the presence of a specific fusion gene. EWSR1-CREM gene fusions have recently been found in several types of tumors. – We wanted to test whether the baseline expression of CREM in normal tissues would be low enough for the detection of overexpression of a fusion protein. The antibody readily detected an EWSR1-CREM fusion protein in Western blotting of a melanoma cell line, and IHC in an MEC with EWSR1-CREM fusion gene was positive. However, so were MECs without a EWSR1-CREM fusion gene as well as most normal tissues. IHC found positive CREM staining in cell nuclei in virtually all tissues and cell types. With this widely positive background, use of CREM staining for detecting EWSR1-CREM fusion protein in tumors is unlikely a feasible approach. In transcriptomic analysis, we found that CREM may be clearly upregulated or downregulated in cancer, depending on the tissue type. This divergence is best explained by the dual role of the CREM gene, as it can act as an enhancer or repressor of transcription, depending on which isoform is expressed. Indeed, isoform-specific expression of CREM has already been linked to prostate cancer; the ICER isoform has been identified as a possible tumor suppressor and CREM a possible coregulator of apoptosis and carcinogenesis with several differentially expressed miRNAs, affecting prostate cancer progression. A study in another cancer type, esophageal squamous cell carcinoma (ESCC), found CREM to have prognostic value. Lower CREM expression was associated with a higher risk of metastasis as well as a poorer response to cisplatin, a treatment used in ESCC among many other cancers. Further studies are warranted to demonstrate the mechanisms behind altered CREM expression, and the consequences thereof, in other forms of cancer. 

sj-pdf-1-jhc-10.1369_00221554211032008 – Supplemental material for Expression of Transcription Factor CREM in Human Tissues Click here for additional data file. Supplemental material, sj-pdf-1-jhc-10.1369_00221554211032008 for Expression of Transcription Factor CREM in Human Tissues by Heidi Kaprio, Vanina D. Heuser, Katri Orte, Mikko Tukiainen, Ilmo Leivo and Maria Gardberg in Journal of Histochemistry & Cytochemistry 

