Long-Term Survival of a Patient With Anaplastic Thyroid Carcinoma Treated With Hypofractionated Radiotherapy: A Case Report.

Anaplastic thyroid carcinoma, a rare type of primary thyroid cancer, is one of the most aggressive neoplasms with a poor prognosis. Many cases are in the advanced stage at the time of the initial visit, and curative treatment is impossible. Because of the highly radioresistant nature of anaplastic thyroid carcinoma, this condition cannot be properly controlled with conventional radiotherapy. Herein, we report the case of a patient with anaplastic thyroid carcinoma who underwent hypofractionated radiotherapy, attained a complete response, and is still alive more than 10 years after treatment with no evidence of disease. To overcome the high radioresistance of anaplastic thyroid carcinoma, we administered 50 Gy in 10 fractions three times a week. Furthermore, we administered paclitaxel and carboplatin sequentially before and after radiotherapy. Consequently, the patient completed treatment and reached a complete response. He is still alive more than 10 years after treatment with no evidence of disease or severe adverse events. Hypofractionated radiation therapy may provide good control of locally advanced anaplastic thyroid carcinoma.

TGF-ß signaling promotes desmoid tumor formation via CSRP2 upregulation.

Desmoid tumors (DTs), also called desmoid-type fibromatoses, are locally aggressive tumors of mesenchymal origin. In the present study, we developed a novel mouse model of DTs by inducing a local mutation in the Ctnnb1 gene, encoding ß-catenin in PDGFRA-positive stromal cells, by subcutaneous injection of 4-hydroxy-tamoxifen. Tumors in this model resembled histologically clinical samples from DT patients and showed strong phosphorylation of nuclear SMAD2. Knockout of SMAD4 in the model significantly suppressed tumor growth. Proteomic analysis revealed that SMAD4 knockout reduced the level of Cysteine-and-Glycine-Rich Protein 2 (CSRP2) in DTs, and treatment of DT-derived cells with a TGF-ß receptor inhibitor reduced CSRP2 RNA levels. Knockdown of CSRP2 in DT cells significantly suppressed their proliferation. These results indicate that the TGF-ß/CSRP2 axis is a potential therapeutic target for DTs downstream of TGF-ß signaling.

Decreased liver B vitamin-related enzymes as a metabolic hallmark of cancer cachexia.

Cancer cachexia is a complex metabolic disorder accounting for ~20% of cancer-related deaths, yet its metabolic landscape remains unexplored. Here, we report a decrease in B vitamin-related liver enzymes as a hallmark of systemic metabolic changes occurring in cancer cachexia. Metabolomics of multiple mouse models highlights cachexia-associated reductions of niacin, vitamin B6, and a glycine-related subset of one-carbon (C1) metabolites in the liver. Integration of proteomics and metabolomics reveals that liver enzymes related to niacin, vitamin B6, and glycine-related C1 enzymes dependent on B vitamins decrease linearly with their associated metabolites, likely reflecting stoichiometric cofactor-enzyme interactions. The decrease of B vitamin-related enzymes is also found to depend on protein abundance and cofactor subtype. These metabolic/proteomic changes and decreased protein malonylation, another cachexia feature identified by protein post-translational modification analysis, are reflected in blood samples from mouse models and gastric cancer patients with cachexia, underscoring the clinical relevance of our findings.

Functional outcome of a patient after hip disarticulation due to an infection 10 years after limb salvage surgery for osteosarcoma: A case report.

Limb salvage is a common procedure after extensive osteosarcoma resection. However, the long-term outcomes after limb salvage surgery (LSS) remain unclear. In this article, the case of a 24-year-old man who underwent hip disarticulation (HD) after an uncontrollable infection is presented. He was previously diagnosed with right distal femoral osteosarcoma and underwent LSS 10 years before disarticulation. Four years after LSS, an uncontrollable infection developed around the endoprosthesis, which eventually prompted HD. The Musculoskeletal Tumor Society (MSTS) functional rating system and the Toronto Extremity Salvage Score were used to compare the subject's activity statuses after LSS and HD. MSTS functional scores were 53.3% after LSS and 60% after HD. Toronto Extremity Salvage Scores were 78.3% after LSS and 95.8% after HD. The subject's emotional acceptance was 3 for LSS and 5 for HD (0 = worst and 5 = best). Both the MSTS and Toronto Extremity Salvage Scores were greater after HD than after LSS. The subject's improved emotional acceptance of the affected limb after HD contributed to the improved functional assessment scores. Alleviation of pain and other disabilities associated with the infection may have contributed to the higher functional scores after the more recent HD surgery. Even if amputation is unavoidable because of complications, high psychological acceptance may prevent a decrease in patient mobility and quality of life after amputation.

L-2hydroxyglutaric acid rewires amino acid metabolism in colorectal cancer via the mTOR-ATF4 axis.

Oncometabolites, such as D/L-2-hydroxyglutarate (2HG), have directly been implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. Here, we showed that the levels of the L-enantiomer of 2HG (L2HG) were specifically increased in colorectal cancer (CRC) tissues and cell lines compared with the D-enantiomer of 2HG (D2HG). In addition, L2HG increased the expression of ATF4 and its target genes by activating the mTOR pathway, which subsequently provided amino acids and improved the survival of CRC cells under serum deprivation. Downregulating the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) and oxoglutarate dehydrogenase (OGDH) increased L2HG levels in CRC, thereby activating mTOR-ATF4 signaling. Furthermore, L2HGDH overexpression reduced L2HG-mediated mTOR-ATF4 signaling under hypoxia, whereas L2HGDH knockdown promoted tumor growth and amino acid metabolism in vivo. Together, these results indicate that L2HG ameliorates nutritional stress by activating the mTOR-ATF4 axis and thus could be a potential therapeutic target for CRC.

Involvement of clusterin expression in the refractory response of pancreatic cancer cells to a MEK inhibitor.

Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.

The cAMP/PKA/CREB and TGFß/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells.

SIGNIFICANCE: This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

Investigation of absolute intra-rater and inter-rater reliabilities during the muscle hardness estimation.

[Purpose] This study aimed to investigate the absolute intra-rater and inter-rater reliabilities during the measurement of muscle hardness, which is used to evaluate physical therapy. Moreover, we examined the effects of using different equipment types and their positioning on the intra-rater and inter-rater reliabilities. [Participants and Methods] Participants of this study comprised 12 healthy adult male individuals. Two experts and two beginners measured the muscle hardness of the lumbar erector spinae and rectus femoris using three types of hardness meters at two positions, including when the muscle was relaxed and stretched. [Results] Intra-rater fixed bias was observed during some measurements by both experts and beginners. Inter-rater fixed bias was observed during measurements by some experts and not the beginners. [Conclusion] In this study, the measurement of muscle hardness demonstrated a need to reconsider the measurement position and acclimation time. These examinations require the consideration of relative and absolute reliabilities.

Assessing the Utility of 18F-Fluorodeoxyglucose Positron Emission Tomography in the Differential Diagnosis Between Spinal Schwannomas and Meningiomas.

Objective The advantage of 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) for the differential diagnosis of schwannoma and meningioma remains unclear. The purpose of this study was to compare the maximum standardized uptake value (SUVmax) with computed tomography (CT) and magnetic resonance imaging (MRI) findings and assess its utility in the differential diagnosis of schwannomas and meningiomas. Methods This study included 42 patients who underwent surgery and had pathological diagnoses of schwannomas (S group) or meningiomas (M group). Multivariate logistic regression analyses were conducted using meningioma prevalence as the dependent variable, and confounders were selected from those with p-values <0.05, including calcification, dural tail sign, tumor volume, and SUVmax at each spinal level as independent variables. Results The SUVmax of the spinal canal type at the level of the cervical vertebrae was significantly higher in the M group (4.6 ± 0.8) than in the S group (2.7 ± 1.4; P = 0.017). Multivariate logistic regression analysis showed that the dural tail sign was significantly associated with differential diagnosis between the S and M groups (odds ratio [OR], 0.851; 95% confidence interval [CI], 0.704-1.031, p<0.001). Conclusions The dural tail sign on MRI, but not the SUVmax of FDG-PET, was the most useful for the differential diagnosis between schwannomas and meningiomas.

MYB mediates downregulation of the colorectal cancer metastasis suppressor heterogeneous nuclear ribonucleoprotein L-like during epithelial-mesenchymal transition.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

Inhibition of Gli2 suppresses tumorigenicity in glioblastoma stem cells derived from a de novo murine brain cancer model.

The prognosis of glioblastoma remains poor despite intensive research efforts. Glioblastoma stem cells (GSCs) contribute to tumorigenesis, invasive capacity, and therapy resistance. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a stem cell marker, is involved in the maintenance of GSCs, although the properties of Lgr5-positive GSCs remain unclear. Here, the Sleeping-Beauty transposon-induced glioblastoma model was used in Lgr5-GFP knock-in mice identify GFP-positive cells in neurosphere cultures from mouse glioblastoma tissues. Global gene expression analysis showed that Gli2 was highly expressed in GFP-positive GSCs. Gli2 knockdown using lentiviral-mediated shRNA downregulated Hedgehog-related and Wnt signaling pathway-related genes, including Lgr5; suppressed tumor cell proliferation and invasion capacity; and induced apoptosis. Pharmacological Gli inhibition with GANT61 suppressed tumor cell proliferation. Silencing Gli2 suppressed the tumorigenicity of GSCs in an orthotopic transplantation model in vivo. These findings suggest that Gli2 affects the Hedgehog and Wnt pathways and plays an important role in GSC maintenance, suggesting Gli2 as a therapeutic target for glioblastoma treatment.

Synthetic lethality between MyD88 loss and mutations in Wnt/ß-catenin pathway in intestinal tumor epithelial cells.

Although the Wnt/ß-catenin pathway plays a central role in the carcinogenesis and maintenance of colorectal cancer (CRC), attempts to target the pathway itself have not been very successful. MyD88, an adaptor protein of the TLR/IL-1ß signaling, has been implicated in the integrity of the intestines as well as in their tumorigenesis. In this study, we aimed to clarify the mechanisms by which epithelial MyD88 contributes to intestinal tumor formation and to address whether MyD88 can be a therapeutic target of CRC. Conditional knockout of MyD88 in intestinal epithelial cells (IECs) reduced tumor formation in Apc+/Δ716 mice, accompanied by decreased proliferation and enhanced apoptosis of tumor epithelial cells. Mechanistically, the MyD88 loss caused inactivation of the JNK-mTORC1, NF-κB, and Wnt/ß-catenin pathways in tumor cells. Induction of MyD88 knockout in the intestinal tumor-derived organoids, but not in the normal IEC-derived organoids, induced apoptosis and reduced their growth. Treatment with the MyD88 inhibitor ST2825 also suppressed the growth of the intestinal tumor-derived organoids. Knockdown of MYD88 in human CRC cell lines with mutations in APC or CTNNB1 induced apoptosis and reduced their proliferation as well. These results indicate that MyD88 loss is synthetic lethal with mutational activation of the Wnt/ß-catenin signaling in intestinal tumor epithelial cells. Inhibition of MyD88 signaling can thus be a novel therapeutic strategy for familial adenomatous polyposis (FAP) as well as for colorectal cancer harboring mutations in the Wnt/ß-catenin signaling.

[Basic Science of Cancer Cachexia].

Cachexia is considered as a complex metabolic disease accompanied by systemic inflammation. However, we still do not understand the essential nature of the metabolic disorder associated with cachexia or the precise molecular mechanisms that drive cachexia. This reviewsummarizes the current knowledge on the pathogenesis of cancer cachexia obtained mainly from mouse models with emphasis on the findings that could reach the bedside in the future. Basic studies using animal models of cancer cachexia indicate that mediators such as pro-inflammatory cytokines and members of TGF-b superfamily disturb the cross-talks among metabolism-related organs including skeletal muscle, adipose tissue, liver, and CNS and thereby induce the collapse of metabolic homeostasis. The inhibitors of these mediators are currently under development for the treatment of cancer cachexia. Skeletal muscle atrophy is a key feature of cancer cachexia and is induced by enhanced proteolysis via ubiquitin-proteasome system and autophagy-lysosome system, as well as by decreased protein synthesis and increased fatty acid oxidation. Adipose tissue atrophy due to excessive lipolysis is another common feature of cancer cachexia, and the involvement of the browning of white adipose tissue and of the increased energy expenditure associated with the futile cycle of lipolysis/lipogenesis is suggested. The liver of cachectic tumor-bearing mice shows increased gluconeogenesis which leads to energy expenditure via futile cycle, and also develops steatosis due to decreased triglyceride usage. In the CNS, inflammation in the hypothalamus induces anorexia and excessive peripheral energy expenditure. Cachectic animals also showresistance to the appetite-promoting effects of ghrelin. We hope that cancer cachexia will gain better awareness in the near future, leading to the growth and progress of the research field, and that the elucidation of its pathogenesis will contribute to the development of novel preventive/therapeutics strategies.

Stromal iodothyronine deiodinase 2 (DIO2) promotes the growth of intestinal tumors in ApcΔ716 mutant mice.

Iodothyronine deiodinase 2 (DIO2) converts the prohormone thyroxine (T4) to bioactive T3 in peripheral tissues and thereby regulates local thyroid hormone (TH) levels. Although epidemiologic studies suggest the contribution of TH to the progression of colorectal cancer (CRC), the role of DIO2 in CRC remains elusive. Here we show that Dio2 is highly expressed in intestinal polyps of ApcΔ716 mice, a mouse model of familial adenomatous polyposis and early stage sporadic CRC. Laser capture microdissection and in situ hybridization analysis show almost exclusive expression of Dio2 in the stroma of ApcΔ716 polyps in the proximity of the COX-2-positive areas. Treatment with iopanoic acid, a deiodinase inhibitor, or chemical thyroidectomy suppresses tumor formation in ApcΔ716 mice, accompanied by reduced tumor cell proliferation and angiogenesis. Dio2 expression in ApcΔ716 polyps is strongly suppressed by treatment with the COX-2 inhibitor meloxicam. Analysis of The Cancer Genome Atlas data shows upregulation of DIO2 in CRC clinical samples and a close association of its expression pattern with the stromal component, consistently with almost exclusive expression of DIO2 in the stroma of human CRC as revealed by in situ hybridization. These results indicate essential roles of stromal DIO2 and thyroid hormone signaling in promoting the growth of intestinal tumors.

TAZ activation by Hippo pathway dysregulation induces cytokine gene expression and promotes mesothelial cell transformation.

Malignant mesothelioma (MM) constitutes a very aggressive tumor that is caused by asbestos exposure after long latency. The NF2 tumor suppressor gene is mutated in 40-50% of MM; moreover, one of its downstream signaling cascades, the Hippo signaling pathway, is also frequently inactivated in MM cells. Although the YAP transcriptional coactivator, which is regulated by the Hippo pathway, can function as a pro-oncogenic protein, the role of TAZ, a paralog of YAP, in MM cells has not yet been clarified. Here, we show that TAZ is expressed and underphosphorylated (activated) in the majority of MM cells compared to immortalized mesothelial cells. ShRNA-mediated TAZ knockdown highly suppressed cell proliferation, anchorage-independent growth, cell motility, and invasion in MM cells harboring activated TAZ. Conversely, transduction of an activated form of TAZ in immortalized mesothelial cells enhanced these in vitro phenotypes and conferred tumorigenicity in vivo. Microarray analysis determined that activated TAZ most significantly enhanced the transcription of genes related to "cytokine-cytokine receptor interaction." Among selected cytokines, we found that IL-1 signaling activation plays a major role in proliferation in TAZ-activated MM cells. Both IL1B knockdown and an IL-1 receptor antagonist significantly suppressed malignant phenotypes of immortalized mesothelial cells and MM cells with activated TAZ. Overall, these results indicate an oncogenic role for TAZ in MMs via transcriptional induction of distinct pro-oncogenic genes including cytokines. Among these, IL-1 signaling appears as one of the most important cascades, thus potentially serving as a target pathway in MM cells harboring Hippo pathway inactivation.

Clinical and in vitro studies of the correlation between MGMT and the effect of streptozocin in pancreatic NET.

PURPOSE: This study aimed to determine the correlation between DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) status and the response to streptozocin in advanced well-differentiated pancreatic neuroendocrine tumors (WD panNETs). METHODS: To test the hypothesis that MGMT deficiency was required for an alkylating drug response, we retrospectively reviewed the response of 13 patients with WD panNETs to alkylating agents in relation to MGMT status. We also studied MGMT expression in streptozocin resistance using panNET cell lines. RESULTS: The cohort included 54% of patients with and 46% without MGMT expression. Among these, 83.3% (5/6) of MGMT-negative cases showed a partial response to streptozocin. In contrast, only 14.2% (1/7) of MGMT-positive cases showed a partial response (P = 0.013). Induced expression of MGMT in BON1 cells (a panNET cell line with undetectable endogenous MGMT) produced streptozocin resistance. Knockdown of MGMT in QGP1 cells, which express MGMT endogenously, did not alter the response to streptozocin. CONCLUSIONS: We observed a relationship between MGMT status and streptozocin response in both patients and cell culture. Despite limited cases examined, high concordance of negative expression of MGMT and response to streptozocin treatment suggest that MGMT expression can be a potential biomarker for this treatment.

Associations among regorafenib concentrations, severe adverse reactions, and ABCG2 and OATP1B1 polymorphisms.

PURPOSE: The ability of predicting severe adverse reactions caused by regorafenib is important. We evaluated regorafenib concentrations for adverse reaction risks and assessed the relevance of laboratory values and gene polymorphisms. METHODS: A total of 28 Japanese cancer patients who were treated with regorafenib were evaluated for the steady state of serum regorafenib concentrations and adverse reactions for 28 days. In addition, we determined the association of regorafenib concentrations with ABCG2 and OATP1B1 polymorphisms, which are regorafenib transporters. RESULTS: Regorafenib concentrations were significantly higher in the group with Grade 2 or higher total bilirubin elevation and thrombocytopenia compared with the group with grades 0 or 1 [3.45 (2.18-7.31) vs. 1.76 (0.26-2.77) µg/mL, P = 0.01 and 3.45 (2.12-7.31) vs. 1.76 (0.26-2.77) µg/mL, P = 0.02, respectively]. A strong association was noted between serum regorafenib concentrations and total bilirubin levels, but the physical and genetic factors predicting regorafenib pharmacokinetics could not be clarified. CONCLUSIONS: Regorafenib concentrations were associated with total bilirubin elevation and thrombocytopenia. Total serum bilirubin could be a useful marker when estimating regorafenib pharmacokinetics.

Acquired JHDM1D-BRAF Fusion Confers Resistance to FGFR Inhibition in FGFR2-Amplified Gastric Cancer.

FGFR2 gene is frequently amplified in gastric cancer. Recently, targeting FGFR2 has drawn attention as a form of gastric cancer therapy, and FGFR-selective inhibitors have shown promising efficacy in clinical studies. Because overcoming acquired resistance is a common problem with molecular targeting drugs, we investigated a resistant mechanism of FGFR inhibitors using the gastric cancer cell line SNU-16, which harbors FGFR2 amplification. We established single-cell clones of FGFR inhibitor-resistant SNU-16 (AZD-R) by continuous exposure to AZD4547, a selective FGFR inhibitor. To screen the genetic alterations acquired in AZD-R, we ran a comparative genomic hybridization assay and found an amplification of Chr7q34 region. The chromosomal breakpoints were located between the 12th and the 13th exon of jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) and between the 3rd and the 4th exon of BRAF We sequenced cDNA of the AZD-R clones and found fusion kinase JHDM1D-BRAF, which has previously been identified in primary ovarian cancer. Because JHDM1D-BRAF fusion lacks a RAS-binding domain, the dimerization of JHDM1D-BRAF was enhanced. A cell growth inhibition assay using MEK inhibitors and RAF-dimer inhibitors indicated the dependence of AZD-R clones for growth on the MAPK pathway. Our data provide a clinical rationale for using a MEK or RAF dimer inhibitor to treat FGFR2-amplified gastric cancer patients who have acquired resistance through the JHDN1D-BRAF fusion. Mol Cancer Ther; 17(10); 2217-25. ©2018 AACR.

HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), an RNA-binding protein that regulates alternative splicing of pre-mRNA, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here, we show that HNRNPLL promotes cell cycle progression and, hence, proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed threefold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3 and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and at protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation-promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 h of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3 and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3 and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNA encoding regulators of DNA replication and promotes colorectal cancer cell proliferation.

HNRNPLL, a newly identified colorectal cancer metastasis suppressor, modulates alternative splicing of CD44 during epithelial-mesenchymal transition.

OBJECTIVE: Despite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or therapy of colon cancer metastasis. DESIGN: CMT93 cells, a murine rectal cancer cell line with poor metastasising activity, were transduced with lentiviral shRNA library and transplanted into the rectum of syngeneic C57BL/6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNA were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNA. RESULTS: The genome-wide shRNA library screen identified Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) encoding a pre-mRNA splicing factor as a candidate metastasis suppressor gene. Knockdown of Hnrnpll enhanced matrigel invasion activity of colon cancer cells in vitro, as well as their metastatic ability in vivo. An RNA-immunoprecipitation analysis showed Hnrnpll-binding to Cd44 pre-mRNAs, and the level of Cd44 variable exon 6 (Cd44v6), a poor prognosis marker of colorectal cancer, was increased by knocking down Hnrnpll. A neutralising Cd44v6 antibody suppressed the matrigel invasion ability induced by Hnrnpll knockdown. HNRNPLL expression was downregulated when colon cancer cells were induced to undergo epithelial-mesenchymal transition (EMT). Immunohistochemistry of clinical samples indicated that colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression. CONCLUSIONS: HNRNPLL is a novel metastasis suppressor of colorectal cancer, and modulates alternative splicing of CD44 during EMT.