High Expression of Casein Kinase 2 Alpha Is Responsible for Enhanced Phosphorylation of DNA Mismatch Repair Protein MLH1 and Increased Tumor Mutation Rates in Colorectal Cancer.

DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.

SPTAN1 Expression Predicts Treatment and Survival Outcomes in Colorectal Cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality. In a cohort of 189 patients with CRC, we recently showed that expression of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1) was lower in advanced metastatic tumours. The aim of the present study was to clarify the association of intratumoural SPTAN1 expression levels with treatment and survival outcomes in patients with CRC. The analysis was based on histologic assessment of SPTAN1 protein levels in our own CRC cohort, and transcriptome data of 573 CRC cases from The Cancer Genome Atlas (TCGA). We first establish that high intratumoural levels of SPTAN1 protein and mRNA associate with favourable survival outcomes in patients with CRC. Next, a response prediction signature applied to the TCGA data reveals a possible link between high SPTAN1 transcript levels and improved patient responses to FOLFOX chemotherapy. Complementary in vitro experiments confirm that SPTAN1 knockdown strains of the colon cancer cell lines HT-29, HCT116 mlh1-2 and Caco-2 are less responsive to FOLFOX chemotherapy compared with SPTAN1-proficient control strains. Taken together, we identify SPTAN1 as a novel prognostic biomarker in CRC and show that SPTAN1 expression levels may predict patient responses to chemotherapy. These investigations illustrate how an affordable, histology-based diagnostic test could directly impact therapeutic decision-making at the bedside.

Expression and secretion of the pro­inflammatory cytokine IL­8 is increased in colorectal cancer cells following the knockdown of non­erythroid spectrin αII.

Non­erythroid spectrin αII (SPTAN1) expression is decreased in ~40% of cases of MLH1­deficient colorectal cancer (CRC). SPTAN1 knockdown reduces cell viability, cellular mobility and cell­cell contact formation, indicating that the SPTAN1 plays an important role in tumour growth, attachment and in regulating the tumour microenvironment. Changes in the tumour microenvironment can affect the immune response. Therefore, in the present study, proteome arrays were used to analyse the expression of 119 different chemokines and soluble receptors in CRC cell lines in which mutL homologue 1 (MLH1) or SPTAN1 were knocked down. The levels of interleukin (IL)­8 were significantly increased in the cells in which SPTAN1 was knocked down, both at the mRNA and protein level. ELISA demonstrated that the cells in which SPTAN1 was knocked down secreted increased quantities of IL­8, and chemotaxis assays revealed the enhanced trafficking of neutrophils, which was induced by media containing higher levels of IL­8. The IL­8 receptors, CRCX1 and CRCX2, were expressed in all the cell lines examined; however, their expression was not directly associated with IL­8 expression. The results of the present study thus demonstrated that CRC cells in which SPTAN1 was knocked down secreted significantly higher levels of IL­8, which in­turn increased the migration of neutrophilic granulocytes. As MLH1­deficient CRC exhibits an increased infiltration of cytotoxic T­cells and is associated with a decreased SPTAN1 expression, it can thus be hypothesized that CRC with a low SPTAN1 expression may release increased quantities of IL­8, resulting in increased immune cell infiltration.

The Role of Nonerythroid Spectrin αII in Cancer.

Nonerythroid spectrin αII (SPTAN1) is an important cytoskeletal protein that ensures vital cellular properties including polarity and cell stabilization. In addition, it is involved in cell adhesion, cell-cell contact, and apoptosis. The detection of altered expression of SPTAN1 in tumors indicates that SPTAN1 might be involved in the development and progression of cancer. SPTAN1 has been described in cancer and therapy response and proposed as a potential marker protein for neoplasia, tumor aggressiveness, and therapeutic efficiency. On one hand, the existing data suggest that overexpression of SPTAN1 in tumor cells reflects neoplastic and tumor promoting activity. On the other hand, nuclear SPTAN1 can have tumor suppressing effects by enabling DNA repair through interaction with DNA repair proteins. Moreover, SPTAN1 cleavage products occur during apoptosis and could serve as markers for the efficacy of cancer therapy. Due to SPTAN1's multifaceted functions and its role in adhesion and migration, SPTAN1 can influence tumor growth and progression in both positive and negative directions depending on its specific regulation. This review summarizes the current knowledge on SPTAN1 in cancer and depicts several mechanisms by which SPTAN1 could impact tumor development and aggressiveness.

Downregulation of SPTAN1 is related to MLH1 deficiency and metastasis in colorectal cancer.

INTRODUCTION: Colorectal cancers (CRCs) deficient in the DNA mismatch repair protein MutL homolog 1 (MLH1) display distinct clinicopathological features and require a different therapeutic approach compared to CRCs with MLH1 proficiency. However, the molecular basis of this fundamental difference remains elusive. Here, we report that MLH1-deficient CRCs exhibit reduced levels of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1), and that tumor progression and metastasis of CRCs correlate with SPTAN1 levels. METHODS AND RESULTS: To investigate the link between MLH1 and SPTAN1 in cancer progression, a cohort of 189 patients with CRC was analyzed by immunohistochemistry. Compared with the surrounding normal mucosa, SPTAN1 expression was reduced in MLH1-deficient CRCs, whereas MLH1-proficient CRCs showed a significant upregulation of SPTAN1. Overall, we identified a strong correlation between MLH1 status and SPTAN1 expression. When comparing TNM classification and SPTAN1 levels, we found higher SPTAN1 levels in stage I CRCs, while stages II to IV showed a gradual reduction of SPTAN1 expression. In addition, SPTAN1 expression was lower in metastatic compared with non-metastatic CRCs. Knockdown of SPTAN1 in CRC cell lines demonstrated decreased cell viability, impaired cellular mobility and reduced cell-cell contact formation, indicating that SPTAN1 plays an important role in cell growth and cell attachment. The observed weakened cell-cell contact of SPTAN1 knockdown cells might indicate that tumor cells expressing low levels of SPTAN1 detach from their primary tumor and metastasize more easily. CONCLUSION: Taken together, we demonstrate that MLH1 deficiency, low SPTAN1 expression, and tumor progression and metastasis are in close relation. We conclude that SPTAN1 is a candidate molecule explaining the tumor progression and metastasis of MLH1-deficient CRCs. The detailed analysis of SPTAN1 is now mandatory to substantiate its relevance and its potential value as a candidate protein for targeted therapy, and as a predictive marker of cancer aggressiveness.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment.

Paediatric and adult soft tissue sarcomas with NTRK1 gene fusions: a subset of spindle cell sarcomas unified by a prominent myopericytic/haemangiopericytic pattern.

Neoplasms with a myopericytomatous pattern represent a morphological spectrum of lesions encompassing myopericytoma of the skin and soft tissue, angioleiomyoma, myofibromatosis/infantile haemangiopericytoma and putative neoplasms reported as malignant myopericytoma. Lack of reproducible phenotypic and genetic features of malignant myopericytic neoplasms have prevented the establishment of myopericytic sarcoma as an acceptable diagnostic category. Following detection of a LMNA-NTRK1 gene fusion in an index case of paediatric haemangiopericytoma-like sarcoma by combined whole-genome and RNA sequencing, we identified three additional sarcomas harbouring NTRK1 gene fusions, termed 'spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern'. The patients were two children aged 11 months and 2 years and two adults aged 51 and 80 years. While the tumours of the adults were strikingly myopericytoma-like, but with clear-cut atypical features, the paediatric cases were more akin to infantile myofibromatosis/haemangiopericytoma. All cases contained numerous thick-walled dysplastic-like vessels with segmental or diffuse nodular myxohyaline myo-intimal proliferations of smooth muscle actin-positive cells, occasionally associated with thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers, including STAT6, were negative. This study showed a novel variant of myo/haemangiopericytic sarcoma with recurrent NTRK1 gene fusions. Given the recent introduction of a novel therapeutic approach targeting NTRK fusion-positive neoplasms, recognition of this rare but likely under-reported sarcoma variant is strongly encouraged.