Research ArticleExperimental Studies

A TGFβ Signaling Inhibitor, SB431542, Inhibits Reovirus-mediated Lysis of Human Hepatocellular Carcinoma Cells in a TGFβ-independent Manner

IKUHO ISHIGAMI, NAOMI SHUWARI, TADATAKA KAMINADE, HIROYUKI MIZUGUCHI and FUMINORI SAKURAI
Anticancer Research May 2021, 41 (5) 2431-2440; DOI: https://doi.org/10.21873/anticanres.15018

Abstract

Background/Aim: Oncolytic reovirus, which is a non-enveloped virus possessing a 10-segmented double-stranded RNA genome, has been anticipated as a novel class of antitumor agent. Hepatocellular carcinoma (HCC) is considered to be a target suitable for reovirus-mediated virotherapy. Transforming growth factor (TGF)-β plays an important role in the pathogenesis of HCC. TGF-β-signaling inhibitors have proceeded to clinical trials as potential antitumor agents for HCC. On the other hand, TGF-β is involved in induction of expression of cathepsins B and L, which are important for reovirus infection. It remains to be examined whether TGF-β signaling inhibitors affect reovirus-mediated lysis of HCC cells. The aim of this study was to evaluate the effects of TGF-β-signaling inhibitors on tumor cell lysis efficiency of reovirus in human HCC cells. Materials and Methods: Reovirus was added to four types of human HCC cell lines pretreated with one of three TGF-β type I receptor inhibitors: SB431542, A-83-01, or galunisertib (LY2157299). Cell viability, virus genome copy numbers, and virus protein expression were evaluated following reovirus infection. Results: SB431542 significantly inhibited reovirus-mediated killing of human HCC cell lines, while A-83-01 and galunisertib did not inhibit. Conclusion: These data indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-β signaling-independent manner.

Key Words:

Oncolytic viruses, which specifically infect tumor cells and induce tumor cell death, have attracted much attention as a new class of antitumor agent. More than 10 oncolytic viruses have been developed so far. Several types of oncolytic viruses have proceeded to clinical trials against various types of tumors and showed promising results. Among the various types of oncolytic viruses, mammalian orthoreovirus type 3 Dearing (hereafter reovirus), which is a non-enveloped virus possessing 10-segmented double-stranded RNA genome, is the most promising oncolytic virus due to its unique properties (1, 2). Reovirus not only exhibits efficient oncolytic activities on a variety of tumor cell lines, but also efficiently induces antitumor immunity. In addition, reovirus can be intravenously administered to patients because of its superior safety profile. Combination therapy using reovirus and antitumor agents have shown promising therapeutic effects on various types of tumors.

Hepatocellular carcinoma (HCC), which is derived from well-differentiated hepatocytes, is the most common primary liver cancer and the third leading cause of cancer-related deaths in the world (3, 4). HCC mainly occurs in patients with chronic liver diseases and cirrhosis; it is difficult to treat due to its rapid development and confers a poor prognosis for survival. Therefore, it is important to develop effective therapeutics for the treatment of HCC. The transforming growth factor (TGF)-β signaling pathway is considered to be a promising therapeutic target in HCC because this pathway is involved in the pathogenesis of HCC and is often activated in HCC (5, 6). TGF-β signaling inhibitors have proceeded to clinical trials in HCC (7).

HCC is an ideal target for reovirus-mediated virotherapy because RAS, which is a key factor for reovirus-mediated oncolysis (8), is often activated in human HCC (9, 10). In addition, reovirus efficiently accumulates in the liver following systemic administration (11). Two lysosome proteases, cathepsins B and L, playing a crucial role in infection with reovirus, are often up-regulated in various types of tumor, including HCC (12, 13). These cathepsins degrade the outer capsid protein of reovirus, yielding infectious subviral particles (ISVPs). ISVPs can disrupt the endosomal/lysosomal membrane and invade the cytosol, resulting in expression of viral proteins and progeny virus production. Tumor cells showing high activity levels of cathepsins B and L are susceptible to reovirus-mediated oncolysis (14, 15).

Expression levels of cathepsins B and L have been demonstrated to be elevated by TGF-β (16, 17), which led us to hypothesize that TGF-β signaling inhibitors might suppress the expression of cathepsins B and L, resulting in inhibition of reovirus infection in human HCC cells. However, it remained to be examined whether TGF-β signaling inhibitors actually consequently inhibit reovirus-mediated killing of human HCC cells.

In this study, we examined the effects of TGF-β signaling inhibitors on reovirus-mediated killing of human HCC cells.

Materials and Methods

Cell lines and reagents. Cells of the human hepatocellular carcinoma cell lines HepG2 (JCRB1054; JCRB Cell Bank, NIBIO, Osaka, Japan), Hep3B, HLE (JCRB0404), PLC/PRF/5 (JCRB0406), and the human non-HCC cell lines HEK293 (a human embryonic kidney cell line), U87 (a human glioblastoma cell line) and Mewo (a human skin melanoma cell line) were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics at 37°C in an atmosphere with 5% CO2. L929 cells were maintained in Joklik’s modified Eagle’s minimal essential medium with 5% fetal bovine serum, 1% L-glutamine, and antibiotics at 37°C in an atmosphere with 5% CO2. The TGF-β type I receptor inhibitor SB431542 was purchased from Tocris Bioscience (Bristol, UK). A-83-01 and galunisertib, which are inhibitors of TGF-β type I receptor, were obtained from Merck (Darmstadt, Germany) and Focus Biomolecules (Plymouth Meeting, PA, USA), respectively. An siRNA against receptor-interacting serine/threonine kinase 2 (RIPK2), which is inhibited by SB431542 via an off-target effect (18), was purchased from Thermo Scientific (Waltham, MA, USA).

Reovirus. Reovirus (kindly provided by Dr. T Etoh and A Nishizono, Oita University, Oita, Japan) was grown in L929 cells and purified by CsCl ultracentrifugation, followed by overnight dialysis, as previously described (14). Biological titers of reovirus were determined by a plaque-forming assay using L929 cells. ISVPs were freshly prepared by treating purified virions with chymotrypsin immediately prior to infection for 15 min at 37°C as previously described (19). All digestions were performed in a buffer containing 200 μg/ml chymotrypsin, 150 mM NaCl, 15 mM MgCl2, and 10 mM Tris-HCl (pH 7.6).

Reovirus-mediated tumor cell lysis. For evaluation of the cell lysis activity of reovirus, all cell lines were seeded at 0.5×104 cells per well in a 96-well plate. On the following day, TGF-β signaling inhibitors were added to the cells at the following concentrations, SB431542: 2 μM and 10 μM; galunisertib: 2 μM and 10 μM; A-83-01: 0.2 μM and 1 μM. Following a 24-h incubation, cells were infected with reovirus and ISVPs at a multiplicity of infection (MOI) of 20. Cell viability was measured at 72 h post-infection using a cell counting kit-8 (Dojindo Laboratories, Kumamoto, Japan).

For the determination of viability of human HCC cells with RIPK2 knockdown, following reovirus infection, HepG2 and Hep3B cells were transfected with RIPK2 siRNA at a final concentration of 20 μM using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). Reovirus was added to RIPK2-knockdown cells at an MOI of 20 at 48 h after siRNA transfection. Cell viability was assessed at 72 h post-infection as described above.

Real-time reverse transcriptase-polymerase chain reaction analysis of the viral genome. HepG2, Hep3B, HLE, and PLC/PRF/5 cells were seeded at 5×104 cells per well in a 24-well plate. On the following day, TGF-β signaling inhibitors were added to the cells at the following concentrations, SB431542: 10 μM; galunisertib: 10 μM; A-83-01: 1 μM. Following a 24-h incubation, cells were infected with reovirus at an MOI of 20. Total RNA, including viral genomic RNA, was isolated from the cells following 72-h infection using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Real-time reverse transcriptase-polymerase chain reaction analysis of the reovirus genome was performed as previously described (14).

Western blotting. For assessment of TGF-β production in the cells, HepG2, Hep3B, HLE, and PLC/PRF/5 cells were lysed in RIPA buffer, followed by western blotting analysis using anti-human TGF-β1 antibody (3C11 at 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a primary antibody. In order to evaluate the phosphorylation levels of SMAD family member 2 (SMAD2), HepG2 cells were seeded at 1×105 cells/well in a 12-well plate. On the following day, cells were incubated with TGF-β signaling inhibitors (10 μM SB431542; 10 μM galunisertib; 1 μM A-83-01) for 24 h. Recombinant human TGF-β (Peprotech, Rocky Hill, NJ, USA) was added to the cells at a final concentration of 10 ng/ml. Following a 1-h incubation, cell lysates were prepared using RIPA buffer. Western blotting was performed using phospho-SMAD2 antibody (138D4, 1:1000) (Cell Signaling Technology, Danvers, MA, USA). For assessment of sigma 3 protein production by reovirus, HepG2, Hep3B, HLE, and PLC/PRF/5 cells were seeded at 2×104 cells per well in a 12-well plate. On the following day, the cells were incubated with TGF-β signaling inhibitors as above for 24 h. Cells were then infected with reovirus at an MOI of 20 for 72 h, following by western blotting analysis of sigma 3 expression using anti-reovirus sigma 3 monoclonal antibody (1:200) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Expression analysis of junction adhesion molecule-A on the cell surface. HepG2, Hep3B, HLE, and PLC/PRF/5 cells were recovered following a 24-h incubation in the presence of SB431542 at 10 μM. Cells were then labeled with mouse monoclonal antibody to junction adhesion molecule-A (JAM-A) (F11; Hycult Biotech, Uden, the Netherlands) or purified mouse IgG1, isotype control (BD Pharmingen, Franklin Lakes, NJ, USA), then incubated with phycoerythrin-conjugated goat anti-mouse IgG secondary antibody (BD Pharmingen). Flow cytometric analysis was performed using a MACS Quant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). The data were analyzed using FlowJo flow cytometry data analysis software (TreeStar, San Carlos, CA, USA).

Cathepsin activity in cells. The activity levels of cathepsins B and L in HepG2 and Hep3B cells treated with TGF-β signaling inhibitors (SB431542: 2 μM and 10 μM; galunisertib: 2 μM and 10 μM; A-83-01: 0.2 μM and 1 μM) for 24 h were measured using an Innozyme Cathepsin B Activity Assay Kit (Calbiochem, San Diego, CA, USA) and Innozyme Cathepsin L Activity Assay Kit (Calbiochem), respectively, according to the manufacturer’s instructions.

Results

TGF-β expression in human HCC cells. In order to examine the expression levels of TGF-β in human HCC cell lines, western blotting analysis was performed. The results showed that all four human HCC cell lines produced detectable levels of TGF-β1 (Figure 1A). Next, in order to examine whether TGF-β signaling inhibitors at the concentrations used in this study significantly inhibited TGF-β signaling, the phosphorylation status of SMAD2 was examined. SB431542, A-83-01, and galunisertib inhibit TGF-β receptor 1 (ALK5), the type 1 receptor serine/threonine kinase activin A receptor type 1B (ALK4) and activin A receptor type 1C (ALK7), respectively (19-21). All three TGF-β signaling inhibitors significantly inhibited phosphorylation of SMAD2 in HepG2 cells (Figure 1B). There were no apparent differences in the SMAD2/3 levels induced by the TGF-β signaling inhibitors. These data indicate that the TGF-β signaling inhibitors significantly inhibited TGF-β signaling at the concentrations used in this study.

Figure 1.
Figure 1.

Inhibition of phosphorylation of SMAD family members SMAD2/3 by transforming growth factor (TGF)-β signaling inhibitors in human hepatocellular carcinoma (HCC) cells. A: TGF-β1 expression in human HCC cells. B: Inhibition of phosphorylation of SMAD2 by TGF-β signaling inhibitors. Cells pretreated with TGF-β signaling inhibitors were incubated with recombinant TGF-β2 (10 ng/ml) for 1 h, followed by western blotting analysis. DMSO: Dimethyl sulfoxide.

Reovirus-mediated lysis of human HCC cells in the presence of TGF-β signaling inhibitors. In order to examine whether treatment with TGF-β signaling inhibitors suppressed reovirus-mediated lysis of human HCC cells, reovirus was added to human HCC cell lines pretreated with TGF-β signaling inhibitors. Neither A-83-01 nor galunisertib promoted or inhibited reovirus-mediated killing of any of the human HCC cell lines tested (Figure 2A). On the other hand, SB431542 at 10 μM significantly restored the viability of all human HCC cell lines following reovirus infection. None of the TGF-β signaling inhibitors inhibited the reovirus-mediated lysis of human non-HCC cell lines, HEK293, U87 and Mewo (Figure 2B). These results indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-β signaling-independent manner.

Figure 2.
Figure 2.

Reovirus-mediated lysis of human hepatocellular carcinoma cells (A) and non-hepatocellular carcinoma cells HEK293, U87, and Mewo cells (B) in the presence of transforming growth factor-β signaling inhibitors. Cells were preincubated with TGF-β signaling inhibitors for 24 h, followed by infection with reovirus at a multiplicity of infection of 20. Cell viabilities were determined after a 72-h infection. SB: 10 μM SB431542; Gal: 10 μM galunisertib; A83: 1 μM A-83-01. DMSO: Dimethyl sulfoxide. The data are expressed as means±SD (n=3-4). ***Significantly different at p<0.001.

Inhibition of reovirus infection by SB431542. Next, in order to examine whether SB431542 inhibited reovirus infection of human HCC cells, reovirus genome copy numbers and virus protein production in the presence of TGF-β signaling inhibitors were examined. SB431542 mediated a more than 30% reduction in the reovirus genome copy numbers in all human HCC cell lines following a 72-h infection (Figure 3A). Sigma 3 protein levels were significantly reduced by SB431542 in HepG2, Hep3B, and HLE cells, although no apparent reduction was observed in SB431542-treated PLC/PRF/5 cells (Figure 3B). Neither A-83-01 nor galunisertib significantly reduced the virus genome copy numbers or sigma 3 protein levels in these cell lines. These data indicate that SB431542 inhibited reovirus infection in human HCC cells.

Figure 3.
Figure 3.

Reovirus genome copy number (A) and virus protein level (B) in human hepatocellular carcinoma cells pretreated with transforming growth factor (TGF)-β signaling inhibitors following a 72-h infection with reovirus. Cells were pretreated with TGF-β signaling inhibitors SB431542 (SB), galunisertib (Gal), A-83-01 (A83) for 24 h, followed by reovirus infection at a multiplicity of infection of 20. Total RNA and cell lysates were recovered 72 h after reovirus infection, followed by real-time reverse transcriptase-polymerase chain reaction analysis (relative to glyceraldehyde 3-phosphate dehydrogenase) and western blotting analysis. DMSO: Dimethyl sulfoxide. The data are expressed as means±SD (n=4). Significantly different at *p<0.05, **p<0.01 and ***p<0.001.

Mechanism of SB431542-mediated inhibition of reovirus infection. In order to reveal the mechanism of SB431542-mediated inhibition of reovirus infection in human HCC cells, we examined the expression levels of JAM-A, which is a primary receptor for reovirus (22), on human HCC cells following treatment with SB431542. No apparent differences in JAM-A expression levels were observed between SB431542-treated and dimethyl sulfoxide-treated cells (Figure 4A). Next, we examined the expression and activity levels of cathepsins B and L following treatment with TGF-β signaling inhibitors. No statistically significant reduction in the expression or activity levels of cathepsin B was observed in HepG2 or Hep3B cells treated with SB431542 (Figure 4B and D). SB431542 slightly reduced the mRNA level of cathepsin L in HepG2 cells (Figure 4C) but the activity level in HepG2 or Hep3B cells was not significantly affected by SB431542 (Figure 4E). Moreover, SB431542 significantly restored cell viability following treatment with ISVPs (Figure 4F). ISVPs mediated efficient tumor cell lysis even when cathepsins B and L were inhibited (15). The SB431542-mediated viability of ISVP-treated cells was not largely different from that in the cells treated with reovirus or SB431542. These data indicate that SB431542 did not significantly reduce the expression or activity levels of cathepsins B or L. These data indicate that SB431542 did not significantly alter the expression levels of JAM-A, or the activity levels of cathepsins B or L in human HCC cells.

Figure 4.
Figure 4.
Figure 4.

Effects of SB431542, an inhibitor of transforming growth factor-β type I receptor, on HepG2 and Hep3B human hepatocellular carcinoma cells. A: Expression of junction adhesion molecule-A (JAM-A), a primary receptor of reovirus, on the cell surface following SB431542 treatment. Cells were incubated with SB431542 at 10 μM for 24 h then mRNA levels of cathepsins B (B) and L (C) (relative to glyceraldehyde 3-phosphate dehydrogenase) were analyzed. Levels of enzymatic activity of cathepsins B (D) and L (E) were determined in HepG2 and Hep3B cells pretreated with TGF-β signaling inhibitors SB431542 (SB), galunisertib (Gal), A-83-01 (A83) for 24 h. F: Infectious subviral particles (ISVP) were added to cells pretreated with SB431542. Cell viability was determined at 72 h following treatment with ISVPs. DMSO: Dimethyl sulfoxide; RFU: relative fluorescence units. The data are expressed as means±SD (n=4). Significantly different at *p<0.05, **p<0.01 and ***p<0.001.

RIPK2 was not involved in SB431542-mediated inhibition of reovirus infection. In order to examine other cellular factors involved in SB431542-mediated inhibition of reovirus infection in human HCC cells, RIPK2 was knocked down by an siRNA against RIPK2. A previous study reported that SB431542 inhibited RIPK2 via an off-target effect (18). In our experiment, an siRNA against RIPK2 mediated more than 95% reduction in RIPK2 mRNA level following transfection (Figure 5A). Reovirus led to similar levels of tumor lysis in HepG2 and Hep3B cells transfected with siRNA against RIPK2 and a control siRNA (Figure 5B). These data indicate that RIPK2 was not largely involved in the SB431542-mediated inhibition of reovirus infection in human HCC cells and that SB431542 inhibited reovirus-mediated lysis of human HCC cells via an unknown mechanism except for inhibition of TGF-β signaling.

Figure 5.
Figure 5.

Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is not involved in SB431542-mediated inhibition of reovirus infection in human hepatocellular carcinoma cells. A: Knockdown efficiency of an siRNA against RIPK2 in HepG2 and Hep3B cells. B: Cell viability of hepatocellular carcinoma cells with RIPK2 knockdown following reovirus infection. Cells were transfected with an siRNA against RIPK2 at 20 μM for 48 h, followed by infection with reovirus at a multiplicity of infection of 20. Cell viabilities were determined at 72 h after reovirus infection. The data are expressed as means±SD (n=4). ***Significantly different at p<0.001.

Discussion

TGF-β is involved in various cellular activities, including tumor malignancy, maintenance of stemness of stem cells, and epithelial and mesenchymal transition. Numerous TGF-β signaling inhibitors have been developed, and have been widely used in basic studies. Among the TGF-β signaling inhibitors developed so far, SB431542 is most widely used. SB431542 is often used for differentiation of stem cells, activation of immune cells, and inhibition of myofibroblast differentiation via inhibition of TGF-β signaling (23-25). This study demonstrated that SB431542, but not A-83-01 or galunisertib, significantly inhibited the reovirus-mediated killing of human HCC cells. The concentrations of SB431542 used in this study have often been used in cultured cells for inhibition of TGF-β signaling (23, 26, 27). SB431542 is a specific inhibitor of ALK5. In addition to ALK5, ALK4 and ALK7 are inhibited by SB431542 (20). A-83-01 and galunisertib also inhibit not only ALK5 but also ALK4 and ALK7 (20, 21). SB431542, A-83-01, and galunisertib interact with the ATP-binding site in ALK5 (28, 29). Our findings indicate that SB431542 inhibited the reovirus-mediated killing of human HCC cells in a TGF-β signaling-independent manner.

SB431542 did not inhibit reovirus-mediated lysis of HEK293, U87, or Mewo cells, suggesting that SB431542-mediated inhibition of reovirus infection occurs only in human HCC cells. The reason for this is unclear, although presumably human HCC cell-specific molecules which are involved in reovirus infection would be inhibited by SB431542.

Reovirus is a highly promising oncolytic virus due to its superior properties. Although reovirus monotherapy exhibited efficient antitumor effects in animal models (30, 31), combination therapies of reovirus and various types of antitumor agents have been carried out and have exhibited promising results in clinical trials (32, 33). When antitumor agents are used in combination with reovirus, we should be vigilant to ensure that the antitumor agents do not inhibit the infection of tumor cells with reovirus. As described above, TGF-β was demonstrated to enhance the expression of cathepsins B and L (16, 17), which are lysosomal proteinases crucial for reovirus infection. These findings led us to hypothesize that TGF-β signaling inhibitors would inhibit reovirus-mediated killing of tumor cells. Contrary to our hypothesis, this study demonstrated that inhibition of TGF-β signaling did not suppress reovirus-mediated killing of tumor cells, at least under the in vitro culture conditions used here. The expressions of cathepsins B and L are regulated by several transcriptional factors other than SMAD2/3 (34, 35). Thus, signaling pathways other than TGF-β/SMAD signaling appear to have mediated the expression of cathepsins B and L in the HCC cell lines used in this study.

It remains to be evaluated whether TGF-β signaling inhibitors inhibit or promote the in vivo antitumor effects of reovirus. The expression and activity levels of cathepsins B and L in tumor cells different under in vitro and in vivo conditions. Alain et al. demonstrated that reovirus efficiently infected tumor cells resistant to reovirus-mediated in vitro oncolysis when tumor cells were subcutaneously transplanted because of elevation in the activity of cathepsins B and L in the subcutaneous tumors, compared with in vitro-cultured tumor cells (15). TGF-β signaling might be more largely involved in the expression and activity of cathepsins B and L in tumor cells under in vivo than under in vitro culture conditions. On the other hand, TGF-β is involved in the suppression of antitumor immunity (36). TGF-β inhibition results in further up-regulation of reovirus-induced activation of antitumor immunity. Previous studies demonstrated efficient activation of antitumor immunity by the combinatorial use of TGF-β inhibitors and oncolytic viruses (37, 38).

In summary, this study demonstrated that SB431542 inhibited reovirus-mediated lysis of human HCC cells via an unknown mechanism in a TGF-β signaling-independent manner. The other TGF-β inhibitors, A-83-01 and galunisertib, did not inhibit reovirus-mediated lysis of human HCC cells. This study suggests that the off-target effects of small-molecule drugs should be borne in mind when they are used in combination therapies with oncolytic viruses.

Acknowledgements

This study was supported by grants-in-aid for Scientific Research (B) and Challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and by a grant from Takeda Science Foundation.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Authors’ Contributions

    II and NS designed and performed the experiments, analyzed the data, and wrote the article. TK performed the experiments. HM supervised the project and reviewed the article. FS conceived and designed the project, performed the experiments, analyzed data, and wrote the article.

  • Conflicts of Interest

    The Authors declare no competing interests.

  • Received March 11, 2021.
  • Revision received April 12, 2021.
  • Accepted April 14, 2021.

References

  1. ↵
    1. Maitra R,
    2. Ghalib MH and
    3. Goel S
    : Reovirus: a targeted therapeutic – progress and potential. Mol Cancer Res 10(12): 1514-1525, 2012. PMID: 23038811. DOI: 10.1158/1541-7786.MCR-12-0157
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Clements D,
    2. Helson E,
    3. Gujar SA and
    4. Lee PW
    : Reovirus in cancer therapy: an evidence-based review. Oncolytic Virother 3: 69-82, 2014. PMID: 27512664. DOI: 10.2147/OV.S51321
    OpenUrlCrossRefPubMed
  3. ↵
    1. Llovet JM,
    2. Kelley RK,
    3. Villanueva A,
    4. Singal AG,
    5. Pikarsky E,
    6. Roayaie S,
    7. Lencioni R,
    8. Koike K,
    9. Zucman-Rossi J and
    10. Finn RS
    : Hepatocellular carcinoma. Nat Rev Dis Primers 7(1): 6, 2021. PMID: 33479224. DOI: 10.1038/s41572-020-00240-3
    OpenUrlCrossRefPubMed
  4. ↵
    1. El-Serag HB and
    2. Rudolph KL
    : Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132(7): 2557-2576, 2007. PMID: 17570226. DOI: 10.1053/j.gastro.2007.04.061
    OpenUrlCrossRefPubMed
  5. ↵
    1. Giannelli G,
    2. Villa E and
    3. Lahn M
    : Transforming growth factor-β as a therapeutic target in hepatocellular carcinoma. Cancer Res 74(7): 1890-1894, 2014. PMID: 24638984. DOI: 10.1158/0008-5472.CAN-14-0243
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chen J,
    2. Gingold JA and
    3. Su X
    : Immunomodulatory TGF-β signaling in hepatocellular carcinoma. Trends Mol Med 25(11): 1010-1023, 2019. PMID: 31353124. DOI: 10.1016/j.molmed.2019.06.007
    OpenUrlCrossRefPubMed
  7. ↵
    1. Ciardiello D,
    2. Elez E,
    3. Tabernero J and
    4. Seoane J
    : Clinical development of therapies targeting TGFβ: current knowledge and future perspectives. Ann Oncol 31(10): 1336-1349, 2020. PMID: 32710930. DOI: 10.1016/j.annonc.2020.07.009
    OpenUrlCrossRefPubMed
  8. ↵
    1. Coffey MC,
    2. Strong JE,
    3. Forsyth PA and
    4. Lee PW
    : Reovirus therapy of tumors with activated Ras pathway. Science 282(5392): 1332-1334, 1998. PMID: 9812900. DOI: 10.1126/science.282.5392.1332
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Newell P,
    2. Toffanin S,
    3. Villanueva A,
    4. Chiang DY,
    5. Minguez B,
    6. Cabellos L,
    7. Savic R,
    8. Hoshida Y,
    9. Lim KH,
    10. Melgar-Lesmes P,
    11. Yea S,
    12. Peix J,
    13. Deniz K,
    14. Fiel MI,
    15. Thung S,
    16. Alsinet C,
    17. Tovar V,
    18. Mazzaferro V,
    19. Bruix J,
    20. Roayaie S,
    21. Schwartz M,
    22. Friedman SL and
    23. Llovet JM
    : Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol 51(4): 725-733, 2009. PMID: 19665249. DOI: 10.1016/j.jhep.2009.03.028
    OpenUrlCrossRefPubMed
  10. ↵
    1. Calvisi DF,
    2. Ladu S,
    3. Gorden A,
    4. Farina M,
    5. Conner EA,
    6. Lee JS,
    7. Factor VM and
    8. Thorgeirsson SS
    : Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 130(4): 1117-1128, 2006. PMID: 16618406. DOI: 10.1053/j.gastro.2006.01.006
    OpenUrlCrossRefPubMed
  11. ↵
    1. Verdin EM,
    2. Maratos-Flier E,
    3. Kahn CR,
    4. Sodoyez JC,
    5. Sodoyez-Goffaux F,
    6. De Vos CJ,
    7. Lynn SP and
    8. Fields BN
    : Visualization of viral clearance in the living animal. Science 236(4800): 439-442, 1987. PMID: 3031817. DOI: 10.1126/science.3031817
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Ruan J,
    2. Zheng H,
    3. Rong X,
    4. Rong X,
    5. Zhang J,
    6. Fang W,
    7. Zhao P and
    8. Luo R
    : Over-expression of cathepsin B in hepatocellular carcinomas predicts poor prognosis of HCC patients. Mol Cancer 15: 17, 2016. PMID: 26896959. DOI: 10.1186/s12943-016-0503-9
    OpenUrlCrossRefPubMed
  13. ↵
    1. Ruan J,
    2. Zheng H,
    3. Fu W,
    4. Zhao P,
    5. Su N and
    6. Luo R
    : Increased expression of cathepsin L: a novel independent prognostic marker of worse outcome in hepatocellular carcinoma patients. PLoS One 9(11): e112136, 2014. PMID: 25384089. DOI: 10.1371/journal.pone.0112136
    OpenUrlCrossRefPubMed
  14. ↵
    1. Terasawa Y,
    2. Hotani T,
    3. Katayama Y,
    4. Tachibana M,
    5. Mizuguchi H and
    6. Sakurai F
    : Activity levels of cathepsins B and L in tumor cells are a biomarker for efficacy of reovirus-mediated tumor cell killing. Cancer Gene Ther 22(4): 188-197, 2015. PMID: 25633482. DOI: 10.1038/cgt.2015.4
    OpenUrlCrossRefPubMed
  15. ↵
    1. Alain T,
    2. Kim TS,
    3. Lun X,
    4. Liacini A,
    5. Schiff LA,
    6. Senger DL and
    7. Forsyth PA
    : Proteolytic disassembly is a critical determinant for reovirus oncolysis. Mol Ther 15(8): 1512-1521, 2007. PMID: 17519890. DOI: 10.1038/sj.mt.6300207
    OpenUrlCrossRefPubMed
  16. ↵
    1. Yin M,
    2. Soikkeli J,
    3. Jahkola T,
    4. Virolainen S,
    5. Saksela O and
    6. Hölttä E
    : TGF-β signaling, activated stromal fibroblasts, and cysteine cathepsins B and L drive the invasive growth of human melanoma cells. Am J Pathol 181(6): 2202-2216, 2012. PMID: 23063511. DOI: 10.1016/j.ajpath.2012.08.027
    OpenUrlCrossRefPubMed
  17. ↵
    1. Zhang Q,
    2. Han M,
    3. Wang W,
    4. Song Y,
    5. Chen G,
    6. Wang Z and
    7. Liang Z
    : Downregulation of cathepsin L suppresses cancer invasion and migration by inhibiting transforming growth factor β mediated epithelial mesenchymal transition. Oncol Rep 33(4): 1851-1859, 2015. PMID: 25632968. DOI: 10.3892/or.2015.3754
    OpenUrlCrossRefPubMed
  18. ↵
    1. Vogt J,
    2. Traynor R and
    3. Sapkota GP
    : The specificities of small molecule inhibitors of the TGFβ and BMP pathways. Cell Signal 23(11): 1831-1842, 2011. PMID: 21740966. DOI: 10.1016/j.cellsig.2011.06.019
    OpenUrlCrossRefPubMed
  19. ↵
    1. Golden JW,
    2. Linke J,
    3. Schmechel S,
    4. Thoemke K and
    5. Schiff LA
    : Addition of exogenous protease facilitates reovirus infection in many restrictive cells. J Virol 76(15): 7430-7443, 2002. PMID: 12097555. DOI: 10.1128/jvi.76.15.7430-7443.2002
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Inman GJ,
    2. Nicolás FJ,
    3. Callahan JF,
    4. Harling JD,
    5. Gaster LM,
    6. Reith AD,
    7. Laping NJ and
    8. Hill CS
    : SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62(1): 65-74, 2002. PMID: 12065756. DOI: 10.1124/mol.62.1.65
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Fields SZ,
    2. Parshad S,
    3. Anne M,
    4. Raftopoulos H,
    5. Alexander MJ,
    6. Sherman ML,
    7. Laadem A,
    8. Sung V and
    9. Terpos E
    : Activin receptor antagonists for cancer-related anemia and bone disease. Expert Opin Investig Drugs 22(1): 87-101, 2013. PMID: 23127248. DOI: 10.1517/13543784.2013.738666
    OpenUrlCrossRefPubMed
  22. ↵
    1. Campbell JA,
    2. Schelling P,
    3. Wetzel JD,
    4. Johnson EM,
    5. Forrest JC,
    6. Wilson GA,
    7. Aurrand-Lions M,
    8. Imhof BA,
    9. Stehle T and
    10. Dermody TS
    : Junctional adhesion molecule A serves as a receptor for prototype and field-isolate strains of mammalian reovirus. J Virol 79(13): 7967-7978, 2005. PMID: 15956543. DOI: 10.1128/JVI.79.13.7967-7978.2005
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Ozawa T,
    2. Takayama K,
    3. Okamoto R,
    4. Negoro R,
    5. Sakurai F,
    6. Tachibana M,
    7. Kawabata K and
    8. Mizuguchi H
    : Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine. Sci Rep 5: 16479, 2015. PMID: 26559489. DOI: 10.1038/srep16479
    OpenUrlCrossRefPubMed
    1. Katsuno Y,
    2. Meyer DS,
    3. Zhang Z,
    4. Shokat KM,
    5. Akhurst RJ,
    6. Miyazono K and
    7. Derynck R
    : Chronic TGF-β exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition. Sci Signal 12(570): eaau8544, 2019. PMID: 30808819. DOI: 10.1126/scisignal.aau8544
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Shaw AK,
    2. Pickup MW,
    3. Chytil A,
    4. Aakre M,
    5. Owens P,
    6. Moses HL and
    7. Novitskiy SV
    : TGFβ signaling in myeloid cells regulates mammary carcinoma cell invasion through fibroblast interactions. PLoS One 10(1): e0117908, 2015. PMID: 25629162. DOI: 10.1371/journal.pone.0117908
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ravindranathan P,
    2. Pasham D,
    3. Balaji U,
    4. Cardenas J,
    5. Gu J,
    6. Toden S and
    7. Goel A
    : A combination of curcumin and oligomeric proanthocyanidins offer superior anti-tumorigenic properties in colorectal cancer. Sci Rep 8(1): 13869, 2018. PMID: 30218018. DOI: 10.1038/s41598-018-32267-8
    OpenUrlCrossRefPubMed
  27. ↵
    1. Tojo M,
    2. Hamashima Y,
    3. Hanyu A,
    4. Kajimoto T,
    5. Saitoh M,
    6. Miyazono K,
    7. Node M and
    8. Imamura T
    : The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta. Cancer Sci 96(11): 791-800, 2005. PMID: 16271073. DOI: 10.1111/j.1349-7006.2005.00103.x
    OpenUrlCrossRefPubMed
  28. ↵
    1. Yingling JM,
    2. McMillen WT,
    3. Yan L,
    4. Huang H,
    5. Sawyer JS,
    6. Graff J,
    7. Clawson DK,
    8. Britt KS,
    9. Anderson BD,
    10. Beight DW,
    11. Desaiah D,
    12. Lahn MM,
    13. Benhadji KA,
    14. Lallena MJ,
    15. Holmgaard RB,
    16. Xu X,
    17. Zhang F,
    18. Manro JR,
    19. Iversen PW,
    20. Iyer CV,
    21. Brekken RA,
    22. Kalos MD and
    23. Driscoll KE
    : Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-β receptor type I inhibitor. Oncotarget 9(6): 6659-6677, 2017. PMID: 29467918. DOI: 10.18632/oncotarget.23795
    OpenUrlCrossRefPubMed
  29. ↵
    1. Ogunjimi AA,
    2. Zeqiraj E,
    3. Ceccarelli DF,
    4. Sicheri F,
    5. Wrana JL and
    6. David L
    : Structural basis for specificity of TGFβ family receptor small molecule inhibitors. Cell Signal 24(2): 476-483, 2012. PMID: 21983015. DOI: 10.1016/j.cellsig.2011.09.027
    OpenUrlCrossRefPubMed
  30. ↵
    1. Katayama Y,
    2. Tachibana M,
    3. Kurisu N,
    4. Oya Y,
    5. Terasawa Y,
    6. Goda H,
    7. Kobiyama K,
    8. Ishii KJ,
    9. Akira S,
    10. Mizuguchi H and
    11. Sakurai F
    : Oncolytic reovirus inhibits immunosuppressive activity of myeloid-derived suppressor cells in a TLR3-dependent manner. J Immunol 200(8): 2987-2999, 2018. PMID: 29555782. DOI: 10.4049/jimmunol.1700435
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Etoh T,
    2. Himeno Y,
    3. Matsumoto T,
    4. Aramaki M,
    5. Kawano K,
    6. Nishizono A and
    7. Kitano S
    : Oncolytic viral therapy for human pancreatic cancer cells by reovirus. Clin Cancer Res 9(3): 1218-1223, 2003. PMID: 12631628.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Harrington KJ,
    2. Vile RG,
    3. Melcher A,
    4. Chester J and
    5. Pandha HS
    : Clinical trials with oncolytic reovirus: moving beyond phase I into combinations with standard therapeutics. Cytokine Growth Factor Rev 21(2-3): 91-98, 2010. PMID: 20223697. DOI: 10.1016/j.cytogfr.2010.02.006
    OpenUrlCrossRefPubMed
  33. ↵
    1. Mahalingam D,
    2. Goel S,
    3. Aparo S,
    4. Patel Arora S,
    5. Noronha N,
    6. Tran H,
    7. Chakrabarty R,
    8. Selvaggi G,
    9. Gutierrez A,
    10. Coffey M,
    11. Nawrocki ST,
    12. Nuovo G and
    13. Mita MM
    : A phase ii study of Pelareorep (REOLYSIN®) in combination with gemcitabine for patients with advanced pancreatic adenocarcinoma. Cancers (Basel) 10(6): 160, 2018. PMID: 29799479. DOI: 10.3390/cancers10060160
    OpenUrlCrossRefPubMed
  34. ↵
    1. Bien S,
    2. Ritter CA,
    3. Gratz M,
    4. Sperker B,
    5. Sonnemann J,
    6. Beck JF and
    7. Kroemer HK
    : Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells. Mol Pharmacol 65(5): 1092-1102, 2004. PMID: 15102937. DOI: 10.1124/mol.65.5.1092
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Yamaguchi T,
    2. Naruishi K,
    3. Arai H,
    4. Nishimura F and
    5. Takashiba S
    : IL-6/sIL-6R enhances cathepsin B and L production via caveolin-1-mediated JNK-AP-1 pathway in human gingival fibroblasts. J Cell Physiol 217(2): 423-432, 2008. PMID: 18543249. DOI: 10.1002/jcp.21517
    OpenUrlCrossRefPubMed
  36. ↵
    1. Batlle E and
    2. Massagué J
    : Transforming Growth Factor-β Signaling in Immunity and Cancer. Immunity 50(4): 924-940, 2019. PMID: 30995507. DOI: 10.1016/j.immuni.2019.03.024
    OpenUrlCrossRefPubMed
  37. ↵
    1. Hutzen B,
    2. Chen CY,
    3. Wang PY,
    4. Sprague L,
    5. Swain HM,
    6. Love J,
    7. Conner J,
    8. Boon L and
    9. Cripe TP
    : TGF-β inhibition improves oncolytic herpes viroimmunotherapy in murine models of rhabdomyosarcoma. Mol Ther Oncolytics 7: 17-26, 2017. PMID: 29034312. DOI: 10.1016/j.omto.2017.09.001
    OpenUrlCrossRefPubMed
  38. ↵
    1. Esaki S,
    2. Nigim F,
    3. Moon E,
    4. Luk S,
    5. Kiyokawa J,
    6. Curry W Jr.,
    7. Cahill DP,
    8. Chi AS,
    9. Iafrate AJ,
    10. Martuza RL,
    11. Rabkin SD and
    12. Wakimoto H
    : Blockade of transforming growth factor-β signaling enhances oncolytic herpes simplex virus efficacy in patient-derived recurrent glioblastoma models. Int J Cancer 141(11): 2348-2358, 2017. PMID: 28801914. DOI: 10.1002/ijc.30929
    OpenUrlCrossRefPubMed
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A TGFβ Signaling Inhibitor, SB431542, Inhibits Reovirus-mediated Lysis of Human Hepatocellular Carcinoma Cells in a TGFβ-independent Manner
IKUHO ISHIGAMI, NAOMI SHUWARI, TADATAKA KAMINADE, HIROYUKI MIZUGUCHI, FUMINORI SAKURAI
Anticancer Research May 2021, 41 (5) 2431-2440; DOI: 10.21873/anticanres.15018
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