Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and ...serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.
Emerging evidence indicates that alternative splicing plays a critical role in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have ...recently attracted considerable attention as a novel class of cancer therapeutics. CDC-like kinases (CLKs) are central to exon recognition in mRNA splicing and CLK inhibitors exhibit anti-tumour activities. Most importantly, molecular mechanism-based combination strategies for cancer therapy must be considered. However, it remains unclear whether CLK inhibitors modulate expression and splicing of apoptosis-related genes, and whether CLK inhibitors enhance cytotoxicity in combination with apoptosis inducers. Here we report an appropriate mechanism-based drug combination approach. Unexpectedly, we found that the CLK inhibitor T3 rapidly induced apoptosis in A2780 cells and G2/M cell cycle arrest in HCT116 cells. Regardless of the different phenotypes of the two cancer cell types, T3 decreased the levels of anti-apoptotic proteins (cIAP1, cIAP2, XIAP, cFLIP and Mcl-1) for a short period of exposure and altered the splicing of the anti-apoptotic MCL1L and CFLAR isoform in A2780 and HCT116 cells. In contrast, other members of the Bcl-2 family (i.e., Bcl-xL and Bcl-2) were resistant to T3-induced expression and splicing modulation. T3 and a Bcl-xL/Bcl-2 inhibitor synergistically induced apoptosis. Taken together, the use of a CLK inhibitor is a novel therapeutic approach to sensitise cancer cells to Bcl-xL/Bcl-2 inhibitors.
Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) ...intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.
Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined by their abilities of tumor initiation, self-renewal and differentiation. In a previous study, we showed by gene knockdown ...using siRNA and gene overexpression experiments that Dnaj (Hsp40) homolog, subfamily B, member 8 (DNAJB8), a role in the maintenance, of renal cell carcinoma CSCs/CICs. In the present study, we established Dnajb8 knockout (KO) renal cell carcinoma (RCC) line cells (RenCa cells) and analyzed the cells to confirm the function of Dnajb8 in RCC CSCs/CICs. Dnajb8 KO cells showed reduced ratios of side population cells and reduced sphere forming ability. An in vivo single cell tumor initiation assay revealed that the numbers of CSCs/CICs were 3 in 4 wild-type RenCa cells and 1 in 4 Dnajb8 KO cells. Dnajb8 KO cells showed sensitivity to Docetaxel. On the other hand, Dnajb8 KO cells did not show any sensitivities to stresses including low pH, low glucose, heat shock and sensitivity to cisplatin. The results indicate that Dnajb8 has a role in tumor initiation, side population ratio and sphere formation but it is dispensable for stress responses.
In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem‐like cells (CSC)/cancer‐initiating cells (CIC) and that it has a role in the ...maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT‐PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem‐like cells.
Cancer stem cells are induced by cellular stress. Cellular stress activates HSF1 and induces DNAJB8, a cancer stem cell‐specific heat shock protein. HSF1/DNAJB8 is a novel pathway of cancer stem cells.
Immune checkpoint inhibitors (ICIs) are used in cancer immunotherapy to block programmed death-1 and cytotoxic T-lymphocyte antigen 4, but the response rate for ICIs is still low and tumor cell ...heterogeneity is considered to be responsible for resistance to immunotherapy. Tumor-infiltrating lymphocytes (TILs) have an essential role in the anti-tumor effect of cancer immunotherapy; however, the specificity of TILs in renal cell carcinoma (RCC) is elusive. In this study, we analyzed a 58-year-old case with clear cell RCC (ccRCC) with the tumor showing macroscopic and microscopic heterogeneity. The tumor was composed of low-grade and high-grade ccRCC. A tumor cell line (1226 RCC cells) and TILs were isolated from the high-grade ccRCC lesion, and a TIL clone recognized a novel neoantigen peptide (YVVPGSPCL) encoded by a missense mutation of the tensin 1 (
TNS1
) gene in a human leukocyte antigen-C*03:03-restricted fashion. The
TNS1
gene mutation was not detected in the low-grade ccRCC lesion and the TIL clone did not recognized low-grade ccRCC cells. The missense mutation of TNS1 encoding the S1309Y mutation was found to be related to cell migration by gene over-expression. These findings suggest that macroscopically and microscopically heterogenous tumors might show heterogenous gene mutations and reactivity to TILs.
The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients ...with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).
Randomized phase II trial of survivin 2B peptide vaccination for patients with HLA‐A24‐positive pancreatic adenocarcinoma is reported. Although peptide vaccination did not improved progression‐free survival, subgroup analysis revealed that longer peptide vaccination might confer survival benefit.
Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides ...derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDH
high
clone cells were established. ALDH
high
clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDH
high
clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDH
high
clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.
Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is ...effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (
CLSPN
) was overexpressed.
CLSPN
mRNA knockdown revealed that
CLSPN
had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted
CLSPN
peptide by HLA ligandome analysis. Thus, we generated a
CLSPN
peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that
CLSPN
is a driver of cisplatin resistance and
CLSPN
peptide-specific immunotherapy may be effective for cisplatin-resistant cases.
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