Cancer-type dependent expression of CK2 transcripts

A multitude of proteins are aberrantly expressed in cancer cells, including the oncogenic serine-threonine kinase CK2. In a previous report, we found increases in CK2 transcript expression that could explain the increased CK2 protein levels found in tumors from lung and bronchus, prostate, breast, colon and rectum, ovarian and pancreatic cancers. We also found that, contrary to the current notions about CK2, some CK2 transcripts were downregulated in several cancers. Here, we investigate all other cancers using Oncomine to determine whether they also display significant CK2 transcript dysregulation. As anticipated from our previous analysis, we found cancers with all CK2 transcripts upregulated (e.g. cervical), and cancers where there was a combination of upregulation and/or downregulation of the CK2 transcripts (e.g. sarcoma). Unexpectedly, we found some cancers with significant downregulation of all CK2 transcripts (e.g. testicular cancer). We also found that, in some cases, CK2 transcript levels were already dysregulated in benign lesions (e.g. Barrett’s esophagus). We also found that CK2 transcript upregulation correlated with lower patient survival in most cases where data was significant. However, there were two cancer types, glioblastoma and renal cell carcinoma, where CK2 transcript upregulation correlated with higher survival. Overall, these data show that the expression levels of CK2 genes is highly variable in cancers and can lead to different patient outcomes.


Introduction
In humans, there are two CK2 kinase genes, CSNK2A1 and CSNK2A2, that code for highlyconserved serine/threonine kinase proteins, CK2α and CK2α', respectively. CK2α and CK2α' differ in their C-terminal sequence [1][2][3][4]. They also differ in their expression pattern and the phenotype of knockout in mice. Thus, CK2α has higher levels and more widespread expression in mouse tissues than CK2α' (mostly brain and testis) [5]. CK2α deficient mice die at mid-gestation while CK2α' deficient mice are viable albeit males are sterile, suggesting that they have different functions that cannot be compensated by the other protein [5,6]. CK2 kinases can function as monomeric kinases, and also within a tetrameric complex composed of two CK2 kinase proteins (CK2α and/or CK2α') and two regulatory proteins with no enzymatic activity (CK2β, coded by CSNK2B). Within this tetrameric complex, CK2β alters CK2 kinase substrate specificity [7]. Additionally, an intronless CK2α pseudogene (CK2aP, coded a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Oncomine analysis
The transcript level of CK2 genes and pseudogene (CSNK2A1, CSNK2A2, CSNK2B, and CSNK 2A1P/CSNK2A3) was analyzed using the Oncomine database (www.oncomine.org, July 2017, Compendia Bioscience, Ann Arbor, MI) [41]. This database contains different datasets, each containing the data from a single publication. We used datasets that compared cancer vs. normal patient specimens for the different cancer types. We used the default view, where all the expression data included is obtained before cancer treatment. Oncomine uses t-test statistics to compare the means of gene expression to determine whether a gene is significantly over or underexpressed in tumors compared to normal tissue. Each Oncomine search provides the p-value (probability that there is a true difference in gene expression), fold change (the difference between the mean

Results and discussion: CK2 transcript expression levels and correlation with overall patient survival by cancer type
Here, the different cancer types studied are organized alphabetically expect for liver cancer, which is placed after all the blood cancers. To simplify the reading, we have chosen to use the terms CK2α, CK2α', CK2β and CK2αP to refer to the genes CSNK2A1, CSNK2A2, CSNK2B, CSNK2A1P/CSNK2A3, respectively.

Bladder cancer
Transitional cell carcinomas (also known as urothelial carcinomas) make up about 90% of all bladder cancers, and originate in cells of the inner lining of the bladder. Transitional cell carcinomas are clinically subdivided into superficial (non-muscle invasive) and invasive tumors. Less common bladder cancer types include squamous cell carcinoma and adenocarcinoma. There is no standard or routine screening test for bladder cancer, leading to a low rate of early diagnosis. Treatments for bladder cancer include surgery, radiation therapy, chemotherapy, and biological therapy.
CK2 in bladder cancer. Oncomine analysis revealed significant overexpression of all three CK2 transcripts in both superficial and invasive types of bladder transitional cell carcinoma (Table 1). In line with our findings, Zhang et al. found overexpression of CK2α transcript in transitional cell carcinomas (subtype not specified) compared with adjacent normal tissue and also in transitional cell carcinoma cells lines compared with normal urinary epithelial cell lines [44]. There were no data in Oncomine for squamous cell carcinoma and adenocarcinoma, possibly due to their low frequency in the population.
Elevated levels of CK2α protein are also found in bladder cancer. Thus, Zhang et al. found increased levels of CK2α protein in bladder carcinomas (subtype not specified) compared with adjacent normal tissue, and in transitional cell carcinoma cells lines compared with normal urinary epithelial cell lines [44]. High staining of CK2α protein is detected in invasive bladder transitional cell carcinoma but not in low grade non-invasive tumors [45]. In addition, CK2α was increased in the lumen of exosomes of metastatic cells versus non-metastatic bladder cancer cells [46]. CK2α protein staining positively correlates with histological grade but not with tumor size, tumor stage or gender [44]. Kaplan-Meier analysis does not reveal an association between high CK2α staining and survival. We further reviewed the role of CK2 proteins in bladder cancer in [16]. All together, these data suggest that CK2α protein, but perhaps not the transcript, could be used as a diagnostic marker in bladder cancer.

Central nervous system (CNS) cancer
Overall (among children and adults), the most common CNS cancer type is glioma (33%), which is subclassified into astrocytomas (20% of total CNS cancer), oligodendrogliomas (2% of total CNS cancer), ependymomas (2% of total CNS cancer), and mixed gliomas. A highgrade (IV) astrocytoma is known as glioblastoma. However, among adults only, the most common CNS cancers is meningioma (33%). Other CNS tumor types include, but are not limited to, medulloblastomas, gangliogliomas, schwannomas, and craniopharyngiomas. While the rate of diagnosis at an early stage is relatively high, with 76.6% of patients diagnosed at the local stage, the 5-year survival for localized CNS cancer is only 36.3%. There are currently no screening tests for CNS cancers. Standard treatments involve watchful waiting, surgery, radiation therapy, chemotherapy, and targeted therapy. CK2 in CNS cancer. Oncomine analysis revealed overexpression and underexpression of CK2α transcripts in astrocytoma and glioblastoma ( Table 2). In agreement with our data, other publications also show under-and over-expression of CK2α transcripts. For example, Zheng et al. show CK2α transcripts mostly overexpressed but also underexpressed in glioblastomas from the TCGA [49]. They attribute increases in CK2α transcripts to gene dosage gains of CSNK2A1. Nitta et al. and Ladha et al. also find increased (mostly) and decreased CK2α transcripts in glioblastomas [38,50]. Dubois et al. find heterogeneous increases in CK2α transcripts in glial brain tumors (oligodendrogliomas, astrocytomas and glioblastoma), but, in this case, CK2 transcript levels did not correlate with gene amplification, suggesting transcriptional mechanisms at play [51]. This heterogeneity in the levels of CK2α transcripts in these cancers may be resolved by increasing the sample size, to determine whether over-or under-expression correlate with any clinicopathological or demographic variables.
Oncomine analysis showed overexpression of CK2α' in oligodendrogliomas and astrocytomas, and over-and under-expression of CK2α' transcripts in glioblastoma. In agreement with  [60] our data, Dubois et al. find elevated levels of CK2α' transcripts in oligodendrogliomas, astrocytomas, and the majority of glioblastomas. They also found CK2α' gene dosage loss in glioblastoma (1/18 samples) [51]. We found overexpression of CK2β transcripts in CNS cancers as detailed in Table 2, except in glioblastoma where there was conflicting data [52,53]. We could not find information on demographic or clinicopathological characteristic that could further explain this discrepancy. Published data also show heterogeneous expression of CK2β transcripts. Thus, some studies find CK2β transcripts mostly unchanged [38] while others find CK2β transcripts mostly upregulated in glial brain tumors (oligodendrogliomas, astrocytomas and glioblastoma) [51]. Further studies will help determine to determine whether over-or under-expression correlate with any clinicopathological or demographic variables. Interestingly, the CK2β gene is deleted in a small percent of glioblastomas (7%, in Zheng et al., and 1/18 in Dubois et al.) [49] Dubois, 2016 #532], but gene dosage gains were also found in a small percent of glioblastomas (1/18 samples) [51]. If after further confirmation, if CK2α' and/or CK2β are found to be downregulated in some subtypes of glioblastoma (grade IV astrocytoma), they could have diagnostic value for these subtypes and also to distinguishing high from low grade astrocytomas that have elevated levels of both transcripts. For other CNS cancer types, Oncomine analysis showed no significant findings for any of the CK2 transcripts. There was no data for CK2αP in astrocytoma.
Regarding CK2 proteins in CNS cancer, Dixit et al. (n = 5) find increased levels of CK2α protein in glioblastomas (5/5 samples), while Nitta et al. find increased (4/7 samples) and decreased (1/7 samples) levels CK2α protein [38,54]. There was a correlation between increased levels of CK2α protein and transcript levels in glioblastomas [38]. In addition, CK2 kinase activity as also higher in most glial brain tumors (oligodendrogliomas, astrocytomas and glioblastoma) but did not correlate neither with transcript levels nor gene amplification [51]. These data suggest that translational regulatory mechanisms, post-translational modifications or different levels of CK2β protein (not measured in these studies) could play a role in the increased CK2 activity observed.
The increase in CK2 activity found in glial brain tumors does not correlate with tumor grade (II, III or IV), and, in the case of glioblastomas, with tumor subtype [51]. However, when CK2 subcellular localization is analyzed, there is a correlation between increased cytoplasmic staining of CK2α with increasing grades of malignancy (grades II, II and IV (glioblastoma)), suggesting that cytoplasmic CK2α protein level has diagnostic value [50]. In contrast, the same study find astrocytomas of all grades show a decrease in nuclear CK2α protein staining compared to control samples. However, other publications show increased levels of both nuclear and cytoplasmic CK2α in CNS cancer. For example, glioblastoma samples show higher staining of CK2α either in the cytoplasm or in both cytoplasm and nucleus, compared with normal brain tissue [51]; and CK2α protein is elevated in cytoplasm and nucleus in grade II, III and IV gliomas (no subtype information) [55]. As for CK2 transcripts, further studies are needed to determine whether over-or under-expression of CK2α proteins correlate with any clinicopathological or demographic variables.
Unexpectedly, Kaplan-Meier analysis showed high expression of CK2α transcripts directly associated with higher overall survival in glioblastoma (p = 0.0235) (Fig 1). In contrast with our analysis, Nitta et al. show high expression of CK2α transcripts correlating with lower survival in mesenchymal glioblastoma, but not in the other types of glioblastomas (classical, neural and proneural) using the TCGA database [38]. In addition, using the Repository of Molecular Brain Neoplasia Data (Rembrandt) they found a trend where high CK2α transcript correlated with poor prognosis [38]. We do not have enough information to explain the difference between our analysis and that of Nitta et al. [38]. As for CK2α protein, there is no difference in patient survival between high staining of CK2α in the cytoplasm or in both cytoplasm and nucleus [51], therefore we may not be able to use CK2α protein upregulation as a prognostic factor in glioblastoma.
Mounting pre-clinical evidence suggests that CK2 inhibitors could be effective in glioblastoma [16]. An additional pre-clinical model show significantly improved survival of mice treated with a combination of TMZ (temozolomide, the standard chemotherapy for glioblastoma) and CK2 inhibitor CX-4945 when compared to TMZ alone (p <0.05) This effect occurs only when drugs are administered simultaneously every 6 days [56]. In addition, inhibition of CK2 with CX-4945 and TBB leads to decreased glioblastoma invasion in glioblastoma cell lines [57].

Cervical cancer
Cervical cancer is the 2 nd leading cause of death among women worldwide, and most commonly caused by human papillomavirus (HPV) infection. Most cases of cervical cancer are preventable with the HPV vaccine. Despite well-established screening tests in place, only 46.9% of patients are diagnosed at the localized stage. Standard treatments involve surgery, radiation therapy, and chemotherapy.
CK2 in cervical cancer. Oncomine analysis revealed significant overexpression of all three CK2 genes in cervical cancer (Table 3). Oncomine had no data on CK2αP expression in cervical cancer.
Kaplan-Meier analysis showed that higher expression of CK2αP transcripts directly correlated with lower survival, suggesting its prognostic value in cervical cancer (p = 0.0034) (Fig 2).
Interestingly, CK2 activity is elevated in HPV-immortalized human keratinocytes and cervical and oral carcinoma cell lines compared to parental cell lines and normal cervical and tongue keratinocytes [61]. In HPV-immortalized cell lines, CK2 activity is highest during the G1 phase of the cell cycle [61]. During G1, CK2 seems to phosphorylate and activate the HPV type 18 E7 protein, a transcription factor that induces cellular proliferation by promoting S-phase entry [61,62]. Based on these data, it is plausible that CK2 stimulates HPV-linked cervical cancer by promoting S-phase entry of the cell cycle. Importantly, CK2 inhibitors are being tested in cervical cancer in clinical trials (reviewed in [16]).

Esophageal cancer
The two most common types of esophageal cancer are adenocarcinoma (most common in USA) and squamous cell carcinoma (most common worldwide). Adenocarcinoma forms in glandular cells close to the stomach, and squamous cell carcinoma forms in flat cells of the upper and middle esophagus. Esophageal cancer is associated with aging, and alcohol and tobacco use (only squamous cell carcinoma), and affects more men than women across all racial and ethnic groups. Importantly, the risk for esophageal adenocarcinoma is increased in Barrett's esophagus, a condition characterized by replacement of the esophageal tissue by tissue similar to that of intestinal lining that occurs in individuals with long-term gastroesophageal reflux disease. Procedures to detect or diagnose esophageal cancer include physical examination, upper endoscopy, chest x-ray, and a barium swallow test, but only 20.5% of patients are diagnosed at the local stage. Treatment options include surgery, radiation therapy, chemotherapy, chemoradiation therapy, laser therapy, and electrocoagulation.
CK2 in esophageal cancer. Oncomine analysis showed significant downregulation of all CK2 genes in Barrett's esophagus and esophageal adenocarcinoma (   [66]. They also find elevated CK2β staining in 86% of tumors levels while the rest have none, weak or moderate staining, and CK2β protein levels are elevated in samples. Furthermore, they find that CK2β transcript expression level correlated with cancer stage (I, II and III) therefore, CK2β transcripts could have diagnostic value [66].

Gastric cancer
The most common type of gastric cancer is adenocarcinoma, accounting for 95% of all gastric cancers, and develops from cells forming the mucosa (innermost lining) of the stomach. Other types can include carcinoid tumor and gastrointestinal stromal tumor. Gastric cancer is more common in men than women, and whites have the lowest rates of gastric cancer compared to all other racial/ethnic groups in the USA. A number of factors increase gastric cancer risk including Helicobacter pylori (H. pylori) infection of the stomach, smoking, family history of gastric cancer, high-salt diet or smoked foods, and low intake of fruits and vegetables. There is currently no standard or routine screening test for gastric cancer. Treatments include surgery, chemotherapy, radiation therapy, and chemoradiation.
CK2 in gastric cancer. Oncomine analysis revealed overexpression of CK2α transcripts in all types of gastric cancer found in Oncomine; some types also showed overexpression of CK2αP and CK2β transcripts (  . As for subcellular localization, CK2α protein staining (nuclear and cytoplasmic) is elevated in gastric carcinoma and dysplastic lesions compared to normal mucosa [29], and CK2β staining is also elevated in gastric carcinoma (nuclear and cytoplasmic) [30].
Kaplan-Meier Plotter analysis showed that higher expression of CK2α (p = 0.010) and CK2β (p = 1.6 10 −5 ) transcripts directly correlated with lower survival in gastric cancer patients ( Fig  3). In addition, published results show that overexpression of CK2α protein correlates with poor survival [29], and elevated levels of nuclear CK2β correlated with poor survival in gastric cancer [30]. Therefore, both CK2α and CK2β transcripts and proteins could be prognostic markers for gastric carcinoma.
CK2α protein was found also to be an independent diagnostic indicator for gastric carcinoma, as nuclear levels of CK2α were associated with higher tumor stage, presence of lymph node metastasis, presence of venous invasion and tumor invasion [29]. Additionally, elevated levels of nuclear CK2β correlated with depth of invasion [30]. These data show that increased CK2α and CK2β protein levels can be correlated with the invasive potential of gastric cancer cells. Indeed, CK2 inhibition led to a decreased migration and invasion of gastric epithelial cells and epithelial-to-mesenchymal transition phenotype, a hallmark for metastasis [29,71]. At least two mechanisms are described to contribute to CK2α's association with gastric carcinoma; through phosphorylation and activation of the protein "deleted in breast cancer 1" (DBC1) [29], and through facilitation of H. pylori's effects on migration and invasion [71]. Overall, both CK2α and CK2β transcripts and proteins have prognostic value in gastric cancer, and CK2α and CK2β proteins have diagnostic value.

Head and neck cancer
Head and neck cancer encompasses cancers in the oral cavity, pharynx, larynx, paranasal sinuses and nasal cavity and salivary glands; 90% arise from squamous cells (e.g. tonsillar carcinoma, tongue carcinoma, floor of the mouth carcinoma, and oropharyngeal carcinoma). The two highest risk factors for head and neck cancer are alcohol and tobacco use. Other risk factors include HPV infection (especially HPV-16), Epstein-Barr virus infection (mainly nasopharyngeal cancer), chewing of paan, drinking yerba mate, eating salty foods, poor oral hygiene, exposure to radiation, and industrial exposures. Treatment options include surgery, chemotherapy, targeted therapy, and/or radiation therapy.
CK2 in head and neck cancer. Oncomine analysis showed that CK2α, CK2α' and CK2β transcripts were significantly overexpressed in head and neck cancers ( Table 6). In addition, Bian et al. using the TGCA database find genetic and transcript expression alterations in CK2α (21%), CK2α' (11%) and CK2β (8%) in head and neck squamous cell carcinoma cases (HNSCC). Most are increases in CK2 transcript expression. However, in a small percentage of tumor samples, CK2 transcript levels decreased [73]. Intriguingly, the majority of these tumor samples have alterations (up or downregulation) in the levels of transcripts of only one of the CK2 genes. There was no data for CK2αP in any of the cancer types in Table 6. There were no studies in Oncomine for other types of head and neck cancer.
HNSCC sections show strong nuclear immunostaining of all three CK2 proteins [33, 74], correlating with elevated levels of CK2 activity in nuclear and chromatin fractions compared to cytosol fractions [31][32][33]. HNSCC cell lines also show increased expression levels of all three CK2 proteins [74].
Kaplan-Meier analysis showed that higher expression of CK2α' (p = 0.002) and CK2β (p = 0.0112) transcripts directly correlated with lower survival in head and neck cancer  [63] patients (Fig 4). In line with these data, there is high CK2 activity (nuclear, cytosolic and chromatin bound) in head and neck cancer [31-33], which correlates with disease status and decreased patient survival [31]. Based on the data above, levels of CK2 transcripts and protein activity could be a prognostic marker for HNSCC. Therefore, CK2 could be a target for cancer therapy. Indeed, pre-clinical evidence suggests that CK2 inhibitors could be effective in HNSCC (reviewed in [16]). In addition, a pre-clinical model of feline oral squamous cell carcinoma, thought to be a good model for human head and neck cancer, show promising results when CK2α and CK2α' are targeted with RNAi [75]. CK2 targeting is associated with low toxicity (little adverse events, with weight loss and anorexia being the most common).

Kidney cancer
Types of kidney cancer include renal cell carcinoma (80%) (subdivided into clear cell, papillary, and chromophobe), renal pelvis carcinoma, transitional cell carcinoma (7%), Wilms tumor (develops in children under 5 years of age), and renal sarcoma (including clear cell sarcoma of the kidney, a rare form that occurs in children typically between 1-4 years of age). Renal oncocytoma is a benign renal tumor. Incidence of kidney cancer has been increasing by an average of 1.6% per year over the past decade. Kidney cancer is twice as common in men as in women, and is also more prevalent among African Americans, American Indians, and Alaska natives. Factors contributing to kidney cancer include smoking, obesity, hypertension, and particular conditions that are inherited. There is currently no standard screening test for kidney cancer. However, individuals with increased risk due to inherited conditions can be screened for kidney cancer using computed tomography (CT) and magnetic resonance imaging (MRI). Treatments include surgery, radiation therapy, chemotherapy, biological therapy, and targeted therapy.   [81]; and propose that CK2 activity may be upregulated in these kidney tumors, as increased expression of CK2β results in increased CK2 activity [81]. Unexpectedly, our Kaplan-Meier analysis showed that higher expression of CK2αP (p = 0.0208) directly correlated with higher survival in patients with renal clear cell carcinoma (Fig 5). In addition, Rabjerg et al. find that high CK2α transcript expression and high nuclear CK2α staining correlate with poor overall survival, disease specific survival and progression free survival in patients with renal cell carcinoma [36,39]. In summary, CK2αP, and CK2α transcript and protein have a

Leukemia
Leukemia is a cancer where the bone marrow produces abnormal white blood cells known as leukemia cells, and is one of the most common childhood cancers. Such cells are non-apoptotic, and their expansion can impede proper function of normal white blood cells, red blood cells, and platelets. Leukemia can be divided into four main types, which are acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL, most common in children), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Hairy cell leukemia (HCL) is rare (2% of all leukemia cases). Risk factors include smoking, chemical exposure such as benzene, radiation exposure, history of chemotherapy or radiation therapy, inherited or genetic conditions, blood disorders, and a family history of leukemia. There is currently no standard screening test for leukemia. Treatments include watchful waiting, chemotherapy, targeted therapy, radiation therapy, and stem cell transplant.
CK2 in leukemia. CK2α', CK2β and CK2αP transcripts were overexpressed in ALL. In Tcell ALL (T-ALL) and B-cell ALL (B-ALL) there were conflicting results for CK2α (Table 8). There was also no mention of patient demographics. Furthermore, the Andersson et al. study used adult controls. Together, these could have accounted for the differences in results between the two studies. Published studies find CK2α protein levels and CK2 activity are elevated in primary B-ALL [89] and T-ALL cells [90]. CK2α' protein levels are elevated in primary B-ALL cells [89]. CK2β protein levels elevated in primary T-ALL cells [90].
In AML, Oncomine analysis showed overexpression of CK2α, CK2α' and CK2αP, and conflicting findings for CK2β. Thus, the Andersson et al. study (Sweden) assayed AML samples from pediatric patients, while the Stegmaier et al. study (USA) used adult patients [87,91]. As above, the intrinsic differences between the pediatric and adult population may play a role in the discrepancy between the two studies for CK2β transcript expression levels. In this regard, there are differences in gene abnormalities between pediatric and adult patients in other cancers and diseases. For example, the frequency of prognostic markers among pediatric and adult B-ALL differ, with adult patients harboring more mutations and epigenetic modifications compared to pediatric patients [92]. Similarly, there are pediatric-adult differences in mature B-cell non-Hodgkin lymphoma and Celiac disease [93,94]. These studies indicate that gene expression alterations may also be different among pediatric and adult AML patients, as we see here for CK2β. Similar to our findings, published studies also demonstrate CK2α transcript overexpression in AML cell lines compared to normal hematopoietic cells [95]. As for CK2 proteins, primary AML cells show both increased and decreased levels of CK2α protein and CK2 activity [95,96]. High CK2α protein levels are a prognostic biomarker, as they correlated with low overall survival and decreased disease-free survival in AML patients [96]. However, high levels of CK2α protein do not correlate with clinical variables, including subtype [96].
CLL   [102]. These data suggest that CK2 is regulated both transcriptionally and post-transcriptionally. In HCL, CK2α' was downregulated. Published data show that in Jurkat, a T-ALL cell line, CSNK2A1P has high transcript levels correlating with gene copy number [103].
There was no data for CML in Oncomine. Pre-clinical evidence suggests that CK2 inhibitors could be effective in treating leukemia (reviewed in [16]).

Lymphoma
Lymphoma, a cancer of the lymphocytes, is the leading blood cancer in the USA. Lymphoma can be subdivided into Hodgkin lymphoma (defined by the presence of Reed-Sternberg cells) and non-Hodgkin lymphoma (NHL). NHLs can derive from B-cells (85%), T-cells (<15%) or NK cells (rare). NHLs can be further subdivided into high grade (fast-growing) and low grade (slow-growing).
Types of B-cell NHLs include low-grade lymphomas (follicular lymphoma, mantle cell lymphoma) and high-grade lymphomas (diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, and primary effusion lymphoma). DLBCL can be further subdivided into activated B cell-like DLBCL, which has the lowest survival of all NHL types, and germinal center B celllike DLBCL. The most common high grade NHL is DLBCL; the most common low grade NHL is follicular lymphoma.
T-cell NHL can include anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, and unspecified peripheral T-cell lymphoma, which are all high-grade lymphomas.
Adult T-cell leukemia/lymphoma (ATL) is usually a highly aggressive NHL.
Risk factors for lymphoma include HIV, Epstein-Barr virus, Helicobacter pylori, or human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection, having a weak immune system and age. Both high and low grade NHL are treated with chemotherapy, and, increasingly, biological (stem cell transplant) or targeted therapies.
CK2 in non-Hodgkin lymphoma. Oncomine analysis showed mixed regulation of all three CK2 transcripts in NHL (Table 9). There was only data for CK2αP in follicular lymphoma and DLBCL. The data in Oncomine for Hodgkin lymphoma was not-significant.
In  With respect to protein levels, CK2α and CK2β protein staining is stronger in follicular lymphoma, Burkitt's lymphoma, and DLBCL, and in lymphoma cell lines, showing both nuclear and cytoplasmic staining [106]. However, the intensity of staining did not correlate with tumor grade, at least in follicular lymphoma [106]. In addition, CK2α and CK2β staining was mantle cell lymphoma tissue sections [107]. Interestingly, overexpression of CK2α in lymphocytes of transgenic mice results in T cell lymphomas, suggesting an oncogenic role for CK2α [17][18][19].

Monoclonal gammopathies
Myeloma is the 3 rd most common blood cancer and is caused by abnormal plasma cells, called myeloma cells, that build up in the bone marrow and cause damage to the solid bone. Accumulation of myeloma cells leading to macroscopic lesions in multiple bone sites is termed "multiple myeloma". In addition to affecting bones, myeloma can also damage other tissues and organs. Types of myeloma include smoldering myeloma (or asymptomatic myeloma), symptomatic myeloma, and plasma cell leukemia, a rare and aggressive form of myeloma with poor prognosis. Elevated levels of antibodies, known as M proteins, produced by myeloma cells in blood, urine, and organs leads to a condition called monoclonal gammopathy of undetermined significance (MGUS). While this condition can be asymptomatic, it can become associated with other diseases or progress to myeloma or other plasma cell malignancies (eg, lymphoma or amyloidosis). Risk factors for myeloma include older age, history of MGUS or having isolated plasmacytoma of the bone. Demografic (age, race), genetic and environmental factors (e.g. radiation, herbicides) play a role in the development of MGUS. There is currently no standard screening test for myeloma. Myeloma treatments include chemotherapy, targeted therapy, biological therapy, corticosteroid treatment, and stem cell transplant with high-dose chemotherapy. In certain instances, radiation or surgery to repair damaged bones may be needed.
CK2 in monoclonal gammopathies. Oncomine analysis showed significant overexpression of CK2α, CK2α' and CK2β (Table 10). MGUS showed conflicting data for CK2α, where . We cannot explain the discrepancy. There was no data for CK2αP in any of the types listed in Table 10. Interestingly, multiple myeloma primary cells and cell lines had elevated levels of CK2α and CK2β proteins and CK2 activity [116], and CK2α and CK2β staining was darker in multiple myeloma tissue sections [107]. However, in MGUS, CK2α and CK2β staining was no different from normal hematopoietic cells [107]. Therefore, it is plausible that CK2 transcript levels are elevated in myeloma.

Liver cancer
Liver cancer most commonly begins in hepatocytes giving rise to hepatocellular carcinoma (also known as primary liver cancer). Other liver cancer types such as fibrolamellar carcinoma, hemangiosarcoma, and hepatoblastoma are rare. Hepatocellular carcinoma is most commonly caused by cirrhosis of the liver due to alcohol abuse, hepatitis B, hepatitis C, hemochromatosis, steatohepatitis, obesity, and diabetes. There is currently no standard screening test for liver cancer. Treatments include surgery, liver transplant, ablation, embolization, radiation therapy, chemotherapy, and targeted therapy.
CK2 in liver cancer. Oncomine analysis showed significant overexpression of CK2α and CK2β in hepatocellular carcinoma (Table 11). In agreement with our analysis, two other groups find CK2α transcripts are upregulated in hepatocellular carcinoma [40,117]. CK2α transcript levels correlate with increasing tumor grade [40], and with elevated levels of CK2α protein in hepatocellular carcinoma samples [40,117]. Therefore, increases in CK2α protein may be due to CK2α transcript upregulation. As for subcellular localization, CK2α staining is found in membrane and cytoplasm [117].
Kaplan-Meier analysis showed that higher expression of CK2α' (p = 0.0083) directly correlated with lower survival in liver cancer (Fig 6). In addition, patients with elevated CK2α transcript [40] and protein staining [117] had a lower survival rate. These authors find that CK2α transcript overexpression is an independent prognostic factor (survival) [40,117].
All together these data show that, CK2α and CK2α' transcripts and CK2α protein have prognostic value, and CK2α transcript and protein have diagnostic value for hepatocellular carcinoma.
In addition to potential treatment of hepatic tumors with CK2 inhibitors, there is a potential role for CK2 inhibition in preventing steatohepatitis and subsequently associated hepatocellular carcinoma, as the CK2 phosphorylation site in Sirtuin 1 is important in the pathophysiology of obesity and hepatic steatosis [118].

Mesothelioma
Mesothelioma is a rare type of cancer that forms from cells of the mesothelium. Mesothelioma is subdivided based on location of origin into pleural (75%), peritoneal (10-20%), pericardial (1%) and testicular (< 1%). Mesothelioma is also classified according to their compromised cell type into epithelial (50-70%), sarcomatoid (10-20%), and biphasic (mixed epithelial and sarcomatoid; 20-35%). Patients with epithelial mesothelioma have the highest survival rate  due to better response to treatment. Risk factors include exposure to asbestos, even for a short amount of time. Other risk factors include radiation exposure, intrapleural thorium dioxide, inhalation of other fibrous silicates, or inheriting a germline mutation on the BAP1 gene. Following diagnosis, only about 40% of patients survive past the first year. There is no standard screening test for mesothelioma, especially for those who were exposed to asbestos. Treatment options include surgery, radiation therapy, chemotherapy, immunotherapy, and/or intraoperative intraperitoneal chemotherapy. CK2 in mesothelioma. Oncomine analysis showed significant overexpression of CK2α and CK2β transcripts in malignant pleural mesothelioma (Table 12). These findings were consistent with a recently published study, where they also find an overexpression of CK2α transcript, and elevated staining of CK2α protein in malignant pleural mesothelioma [122]. There was no data for CK2αP in mesothelioma.
Kaplan-Meier analysis showed that higher expression of CK2αP (p = 0.01) directly correlated with lower survival in mesothelioma, suggesting its prognostic value in this cancer (Fig 7).

Parathyroid cancer
Parathyroid cancer affects any of the four parathyroid glands. Both benign and malignant parathyroid tumors can lead to hyperparathyroidism, where excess parathyroid hormone (PTH) is produced leading to over-absorption of dietary calcium in the intestines, and movement of stored calcium in the bones into blood. These events ultimately lead to hypercalcemia, where amount of calcium in the blood rises above normal levels. Risk factors for parathyroid adenoma include rare inherited disorders such as familial isolated hyperparathyroidism and multiple endocrine neoplasia type 1 syndrome. Treatment options include minimally invasive surgery to remove the tumor itself or treating the hypercalcemia (gallium nitrate, calcitonin, IV fluids, or drugs that prevent breakdown and reabsorption of bones). Chemotherapy and radiation therapy are not very effective in preventing parathyroid cancer recurrence. Furthermore, radiation therapy can instead increase risks of developing parathyroid adenoma.
CK2 in parathyroid overgrowth. Oncomine analysis showed significant overexpression of CK2α transcript in three types of parathyroid overgrowth (hyperplasia and benign), as well as an overexpression of CK2α' transcript in parathyroid hyperplasia (Table 13). There was no data for CK2αP in any of the types in Table 13.

Thyroid cancer
Thyroid cancer occurs in the thyroid gland, where thyroid follicular cells normally produce thyroid hormone which regulates bodily functions such as body temperature, weight, blood pressure, and heart rate. The thyroid gland also consists of C cells which secrete calcitonin, which is important in calcium homeostasis in the body. There are four types of thyroid cancer, including papillary, follicular, medullary, and anaplastic thyroid cancer. Papillary thyroid cancer is the most common among the four, while anaplastic thyroid cancer remains the most aggressive with the lowest cure rates. Risks for thyroid cancer include genetic conditions (eg,  (Table 14). There was no data for CK2αP.
Guo et al. found higher CK2α transcript levels and protein staining (in particular nuclear staining) in all four thyroid carcinoma subtypes (papillary, follicular, medullary, and anaplastic) [125]. Furthermore, higher nuclear CK2α protein staining was associated with lymph node metastasis and tumor stage and EMT markers, suggesting that CK2α staining can have diagnostic value [125]. This indicated a potential role for targeting CK2α for thyroid cancer treatment.

Sarcoma
Sarcoma develops from abnormal cells of mesenchymal origin. This can include muscle, bone, fat, and/or vascular tissues. There are more than 50 types of sarcoma, but the most common types include liposarcoma (adipose tissue), synovial sarcoma (synovial lining cells in joints), and leiomyosarcoma (smooth muscle). Rhabdomyosarcoma is the most common in children. Risk factors for sarcoma include inherited conditions such as retinoblastoma, Li-Faumeni syndrome, familial adenomatous polyposis, neurofibromatosis, Werner syndrome and tuberous sclerosis, and also chemical and/or radiation exposure. There is no current standard screening test for sarcomas, and they are usually diagnosed using imaging techniques such as CT and MRI scans. Additionally, removal of a tissue sample is needed to confirm the diagnosis. Treatment options include surgery, radiation therapy, and/or chemotherapy. CK2 in sarcoma. Overall, Oncomine analysis revealed significant overexpression of CK2α, and mostly underexpression of CK2α' in sarcoma. There was overexpression of CK2β in pleiomorphic liposarcoma and underexression in dedifferentiated liposarcoma. There were conflicting results for CK2α' in leiomyosarcoma (Table 15). However, leiomyosarcoma samples in the Quade et al. study was obtained specifically from patients with leiomyosarcoma of the uterus. Detwiller et al. obtained samples from patients with leiomyosarcoma in any body region, although they did not specify. Control samples in the Quade et al. study were uterus samples from healthy individuals, while control samples in the Detwiller et al. study were soft tissue samples (not specified) from healthy individuals. Thus, these differences in sample source and type may have contributed to the different findings. There was no data for CK2αP in all types listed in Table 15.
Kaplan-Meier analysis showed that higher expression of CK2α transcript (p = 0.0005) directly correlated with lower survival in sarcoma, suggesting its prognostic value in this cancer type (Fig 8).
Takahashi et al. found overexpression of CK2α and CK2β protein in human osteosarcoma cell lines when compared to normal cells [127]. Furthermore, knockdown of either CK2α or CK2β with siRNA or incubation with CX-4945 led to prevention of osteosarcoma cell proliferation. However, CX-4945 did not inhibit cell proliferation of human mesenchymal stem cells, indicating a role for CK2 in osteosarcoma cell proliferation. In vivo results in xenograft mouse models found that CX-4945 inhibited osteosarcoma growth [127]. Together, these indicate a role for CK2 inhibition in osteosarcoma treatment.

Skin cancer
Skin cancer includes melanoma and non-melanoma skin cancer (basal cell carcinoma and squamous cell carcinoma). Melanoma is the most fatal form of skin cancer and arises from melanocytes. On the other hand, non-melanoma skin cancer usually responds well to treatment and is rarely metastatic. A risk factor for skin cancer is the presence of benign melanocytic skin nevus, more commonly known as moles and freckles. Other risk factors include fair skin, high exposure to natural (sun) or artificial UV light, history of blistering sunburns, and history (family or personal) of melanoma or atypical moles. The current standard screen for melanoma is a visual examination of the skin. Treatments include surgery, chemotherapy, targeted therapy, biological therapy, and radiation therapy. CK2 in melanoma. Oncomine analysis revealed significant overexpression of all three CK2 genes as detailed in the Table below (Table 16). There was no data for CK2αP in any of the types listed in Table 16.
Interestingly, ninety percent of melanomas with CK2α transcript upregulation had mutations in the most prevalent genes mutated in melanoma: BRAF, NRAS and/or NF1 [131]. Moreover, these authors found increased CK2α protein expression in melanoma cell lines when compared with normal human melanocytes [131]. Intriguingly, CK2α staining and transcript levels are elevated in melanomas with NRAS Q61 mutations but not in melanomas with NRAS G12 mutations [132]. These suggest a potential for the use of CK2 inhibitors to treat this cancer. We reviewed the use of CK2 inhibitors in combination with current therapies for melanoma in [16].

Testicular cancer
90% of testicular cancer derive from germ cells, and the remaining 10% derive from supporting cells (Leydig cells, Sertoli cells). Germ cell-related testicular cancer can be subdivided equally into seminomas and non-seminomas. Non-seminoma testicular cancer is subdivided into teratomas, embryonal carcinomas (3-4%), mixed germ cell tumors, and yolk sac tumors. Typically, non-seminoma tumors grow faster, present with an earlier diagnosis age, and have a CK2 gene expression in cancer lower relative 5-year survival rate. In contrast, seminomas have a higher survival rate largely due to the fact that they are highly sensitive to radiation therapy and chemotherapy. Germ cell tumors can also occur outside of the gonads due to defects in embryonic development. There are many risk factors for testicular cancer, including cryptorchidism, Turner syndrome, androgen insensitivity syndrome, Klinefelter's syndrome, low fertility, and family history. Treatment options for testicular cancer include surgery, radiation therapy, and chemotherapy. CK2 in testicular cancer. Oncomine analysis revealed significant downregulation of all three CK2 genes and CK2αP in both seminomatous and non-seminomatous testicular cancer (Table 17). CK2αP was also downregulated in testicular seminoma. There was no data for CK2αP in any of the other types listed in Table 17.
To our knowledge, there is no data on the CK2 protein levels in testicular cancer, or on the mechanism by which CK2 dysregulation could be affecting testicular cancer. However, we have knowledge on CK2 levels in normal testicular tissue and in reproductive organ development. Specifically, CK2α' and CK2β transcript, protein levels and CK2 activity are highest in mouse testicles compared to other mouse tissues [5,136,137]. In mice and Xenopus laevis, CK2 transcript levels are high in germ cells (reviewed in [137]). Importantly, CK2α' is required for male gametogenesis, as knockout of CK2α' mice are infertile and have oligospermia and globozoospermia (i.e. spermatozoa having round heads) [5]. In addition, the natural CK2 inhibitor Emodin causes hypospermatogenesis and apoptosis of mouse germ cells [138]. These phenotypes could be due in part to germ cell chromatin breakdown, a risk factor for testicular cancer [139]. Potentially, CK2 could be acting on two chromatin remodeling proteins, CK2 Target protein 2 (CKT2) and testis-specific protein, Y-encoded (TSPY) [140,141]. Since CK2α' is required for sperm differentiation in mice, downregulation of CK2α' in testicular cancer could delay and/or prevent germ cell differentiation, and possibly divert sperm development towards tumor formation.

Conclusion
Our goal was to determine the extent to which CK2 genes could be significantly up-or downregulated in cancers we did not study in our previous publication [9], and to determine whether the CK2 gene expression levels in these cancers correlated with overall patient survival. Here we show that there is dysregulation of CK2 transcripts in a number of these cancer types that was not reported previously, and that over-and under-expression of CK2 transcripts was cancer-type specific. In addition, the prognostic value of CK2 genes was also cancer-type specific as both over-and under-expression correlated with poor survival. In addition, we have compared our data to published results on CK2 gene and protein expression.
In Table 18, we summarized the analysis from Oncomine and of the literature. In many cases these results are robust, as several studies showed the same up-or down-regulation for the CK2 transcript (e.g. up to 4 studies for CK2α and CK2β increase in hepatocellular carcinoma) ( Table 18). The fact that independent studies showed similar data gives strength to the conclusions reached. As we have previously found in other cancers [9], we found cancers with significant upregulation of all three CK2 genes, such as bladder cancers, and tumors with mixed up-and down-regulation of CK2 transcripts. In solid tumors with mixed up-and down-regulation, CK2α' was almost always downregulated (e.g. sarcomas and melanoma), as it was in breast, ovarian and pancreatic cancers [9]. In addition, we found a third pattern of dysregulation of CK2 genes: significant downregulation of all three CK2 transcripts. Finding all three CK2 genes downregulated (e.g. Barrett's esophagus and testicular cancer) was unexpected, particularly since CK2α/α' downregulation leads to decreased proliferation (reviewed Table 17. Analysis of changes in CK2 gene expression in testicular cancer. P-values, fold change, rank and datasets are shown. Not Otherwise Specified was a category in Oncomine however, in the case of Seminoma and teratoma they are the same as the category above. in [16,22,23]). This is a novel finding in CK2α/α' biology, and requires further study to determine whether CK2α/α' protein levels are equally decreased and, if so, which hallmark of cancer [145] could be affected by CK2α' protein decrease. In contrast to the little data on CK2α' downregulation, CK2β downregulation is already linked to cancer progression. In particular, to increased epithelial to mesenchymal transition [146]. Further investigation as to whether CK2 gene downregulation contributes to cancer development in these cancer types, including animal model studies, will address the relevance of this finding. One possibility in testicular cancer is that aberrant expression of CK2α' and CK2β could cause a dysfunction in gametogenesis leading to subfertility; as subfertility has been linked to increased risk of developing testicular cancer [139]. The Oncomine analysis led to additional insights. CK2αP was upregulated or downregulated following the prevalent pattern of up-or down-regulation of the other CK2 transcripts. Similarly, CK2αP follows the same pattern in lung, breast and colorectal cancers [9]. The importance of CK2αP deregulation in cancer is to be determined. We found altered expression of CK2 transcripts already in benign lesions (e.g. parathyroid overgrowth and benign Melanocytic Skin Nevus), and also in Barrett's esophagus (that increases the risk of developing esophageal cancer). This is similar to what is previously found in benign colon adenomas [9]. This suggests a role in cancer initiation in these cancer types. In NHL, low grade (slow-growing) lymphomas (e.g. FL) had downregulation of CK2 transcripts while in high grade NHL (more aggressive) there was upregulation of CK2 transcripts. This suggest differential transactivation of the CK2 genes or differential stability of the transcripts may be playing a role in high grade NHL.
In Table 18 we also summarized the literature on CK2 protein levels in diverse cancers. In general, the increases in CK2 transcripts paralleled increases in CK2 protein expression levels, nuclear localization, and/or activity; with the exception of B-cell ALL, AML, CLL and Follicular lymphoma (Table 18). Therefore, we cannot directly infer the levels of CK2 proteins in human cancer from studies of transcript levels, particularly in blood cancers. These data support the notion that there are multiple layers of regulation of CK2 expression that need to be addressed in future studies.
A number of studies show that higher CK2 protein levels, nuclear localization, and/or activity have diagnostic value, that is, correlate with clinicopathological factors (mostly with higher tumor grade and tumor stage; and sometimes with metastasis and invasion) ( Table 18, asterisks). Only one exception, were increase CK2α' transcript correlated with low grade clear cell renal cell carcinoma Rabjerg, Guerra et al. 2017). The significance of this finding needs to be addressed. Interestingly, the diagnostic value of CK2 proteins is linked mostly to higher overall staining, nuclear staining or CK2 activity. However, in the case of astrocytomas, it is cytoplasmic CK2 staining that has a diagnostic value [50]. Importantly, in some cancers, increases in both CK2 transcript and protein expression have diagnostic value (e.g. Clear Cell Renal Cell Carcinoma [36]) ( Table 18, asterisks). This suggests that both CK2 protein and transcript levels could be used as diagnostic biomarkers. Future in depth analyses will determine the extent to which CK2 transcript and protein levels correlate and have diagnostic value in these cancers. Ultimately, changes in the overall and relative levels of CK2 proteins and of the composition of the CK2 heterotetramer will affect diverse cellular functions (e.g. proliferation, epithelial to mesenchymal transition). These effects could be mediated through changes in the activation of CK2-dependent signaling pathways or expression of downstream genes as reviewed in [16].
In Table 19, we summarized the value of CK2 as a prognostic marker. In most cancers, overexpression of one or two CK2 gene transcripts led to lower survival (e.g. gastric cancer, sarcoma, head and neck and liver cancer) (Table 19). In addition, overexpression of the pseudogene CK2αP led to lower survival in cervical cancer and mesothelioma. However, similar to CK2 gene expression in cancer lung adenocarcinoma [9], overexpression of CK2αP in renal clear cell carcinoma led to increased survival. The significance of this finding is unclear, as we know little about the function of CK2αP. Importantly, there was a tight parallel between the prognostic value of CK2 transcripts and proteins, furthering the notion that abnormal levels of CK2 can be a useful prognostic marker (Table 18). Intriguingly, the levels of CK2α' were not statistically significant in the Oncomine analysis while they were a prognostic marker in liver cancer. This lack of significance in Oncomine analysis may be due to a high standard deviation in CK2α' transcript levels in liver cancer samples. This result indicates that the prognostic value of a CK2 transcript (and perhaps of CK2 protein) needs to be investigated independently of whether the average expression level is significantly different or not from control samples. In this study, as we did for [9], we have followed an unbiased approach to analyze and report all the data available to date in the databases. This has resulted in unique studies which show that both CK2 over-and under-expression have significance in cancer. There are intrinsic limitations to the database analyses. For example, not all cancer types are included in the databases searched, and, in some cases, the number of patients studied is not large enough to find significant differences in transcript levels or in survival rates. There are also inherent factors (algorithms used in microarray analysis, reference genes used for normalization, the age of the samples, preservation, etc) which could limit the outcome of the analyses or contribute to the discrepancies between studies. Those discrepancies should be resolved when additional independent studies are performed. A broader limitation to our study is that Oncomine does not include information on CK2 gene mutations which might also affect overall patient survival.
There is a need for cancer biomarkers (diagnostic, prognostic and predictive), and for therapeutic targets and treatments to address cancer incidence and mortality in the world Table 19. Prognostic value of CK2 transcript and protein in cancer. High CK2 transcript correlate with higher (é) or lower (ê) patient survival. We also included published data on correlation of high CK2 transcripts and protein (indicated as (p)) levels with lower survival (light blue arrow, ê). Each arrow indicates an independent study. (#) = increased CK2 activity correlates with decreased patient survival [31].  (Table 20). Our study advances the notion that CK2 transcript expression is a potential diagnostic and prognostic biomarker for specific types of cancer. However, a number of validation steps are needed before CK2 gene expression has an impact in clinical practice [147][148][149]. This includes whether CK2 transcripts and proteins could be used alone or in combination as diagnostic and prognostic factors. In addition, future studies could begin to address the predictive value of CK2 to select those patients who are more likely to benefit from a particular cancer treatment.