CONCLUSIONS BACKGROUND Targeting stearoyl CoA desaturase 1 (SCD1) in hepatocellular carcinoma Ilah Bok 1 , Laura A. Marlow 1 , James L. Miller 1 , Jason Hall 1 , Akiko Matsuda 2 , Yan Asmann 3 , Catherine D. Moser 4 , Vivekananda Sarangi 5 , Steven R. Alberts 6 , Kabir Mody 7 , Lewis R. Roberts 4 , Mark J. Truty 8 , Tushar C. Patel 2 , John A. Copland 1 1 Department of Cancer Biology, 2 Gastroenterology and Hepatology Department, 3 Health Science Research, 7 Hematology Oncology, Mayo Clinic Florida, Jacksonville, Florida; 4 Gastroenterology; 5 Health Science Research, 6 Medical Oncology, 8 General and GI Surgery, Mayo Clinic, Rochester, MN Abstract 192 / 21 April 2, 2017 1 – 5 pm FUTURE DIRECTIONS RESULTS REFERENCES & ACKNOWLEDGEMENTS Fig. 1. A novel SCD1 inhibitor demonstrated SCD1 specificity and excellent oral bioavailability. Figure 4: SSI-4 inhibits HCC cell proliferation, demonstrating synergy with sorafenib. Objectives: Objectives include demonstration antitumor activity of the lead SCD1 inhibitor, SSI-4, against HCC in cell culture and animal models for HCC. HCC is a Wnt driven cancer. SCD1 mechanisms of action are examined that include Wnt regulation and endoplasmic reticulum (ER) stress. SSI-4 was examined for synergy with FDA approved drugs for HCC such as sorafenib. Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models. Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing (Data not shown). SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins. Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC. Results: 1. We developed novel, high affinity and specific small molecule SCD1 inhibitors through a combined computational and synthetic chemistry approach. 2. SSI-4, the lead SCD1 inhibitor, possesses excellent bio-availibility and can be taken orally with a half-life of 3-4 hours. 3. SSI-4 possesses single agent antitumor activity in HCC PDX mouse models. 4. Soft agar growth predicts SCD1 responsive HCC cell lines. 5. In cell culture, SSI-4 demonstrates anti-proliferative synergy with sorafenib. Conclusions: • Targeting fatty acid metabolism may be a novel therapeutic strategy in treating HCC. • Identify additional combination therapies that may provide antitumor synergy with SCD1 inhibitors. • Demonstrate in vivo antitumor synergy with sorafenib and other novel combination therapies. • Develop our novel lead SCD1 inhibitor, SSI-4, for IND filing and FDA approval towards clinical trials. Figure 4. SCD1 inhibits HCC cell proliferation and demonstrates synergy with sorafenib. A. Dose response of 0.001 – 10 nM SSI-4 with 5 day exposure demonstrates IC50 of 1-3 nM in HCC responsive cell lines. B. Single cells grown in 3D soft agar identified SCD1 sensitive cell lines. C. Sorafenib dose out with same conditions as SSI-4 showed proliferation inhibition for all HCC cell lines. IC50 values are shown. D. Using the method of Chou and Talalay, combination index (CI) showed synergy with combined SSI-4 and sorafenib in HLF cells. Figure 2: SCD1 is overexpressed in HCC and linked to Wnt regulation in HCC Fig. 3. SSI-4 inhibits in vivo tumor growth in HCC patient derived xenograft (PDX) mouse models The paucity of effective therapeutic agents for hepatocellular carcinoma (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor against HCC. ABSTRACT A. Structure of SSI-4 and SSI-2. B. SCD1 inhibitor candidates were evaluated for their inhibition of oleoyl CoA biosynthesis by mouse liver microsomes in vitro. SSI-2 or SSI-4 were incubated with microsomes and 13 C 18 - labeled stearoyl CoA, a native substrate of SCD1. Bioconversion of this substrate to 13 C 18 -labeled oleoyl CoA product was quantified by LC/MS for drug treatments relative to vehicle (DMSO) controls. MF-438, a commercially available SCD1 inhibitor, is used as a comparator. SSI-1 and SSI-3 IC50 values were >1 uM (data not shown). C. HCC cell lines demonstrate inhibition of proliferation with 1 μM SSI-4. To demonstrate SCD1 specificity, cells were pretreated with 5 μM of oleic acid to rescue the growth inhibitory phenotype. Doubling times predict response to SSI-4 inhibition. D. A single dose of oral (PO) or intravenous (iv) SSI-4 was administered to fasted male C57BL/6 mice with plasma levels measured over 24 hours. E. Half-life and bioavailability are key parameters measured in the single dose analyses. Bioavailability (%) was calculated with AUC 0-last and nominal dose. . 1. Nile AH, Hannoush RN. Fatty acylation of Wnt proteins. Nat Chem Biol. 2016;12(2):60-9. 2. Cassidy JW, Caldas C, Bruna A. Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts. Cancer Research. 2015 August 1, 2015;75(15):2963-8. 3. Powers T. Cell Growth Control: mTOR Takes on Fat. Molecular Cell. 2008;31(6):775-6. 4. Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer. 2007;7(10):763-77. 5. Porstmann T, Griffiths B, Chung Y-L, Delpuech O, Griffiths JR, Downward J, Schulze A. PKB//Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP. Oncogene. 2005;24(43):6465-81. 6. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434(7035):843-50. 7. von Roemeling C, Laura A. Marlow L, Pinkerton A, Tun H, Smallridge R, Copland J. Aberrant Lipid Metabolism in Anaplastic Thyroid Carcinoma Reveals Stearoyl CoA Desaturase 1 as a Novel Therapeutic Target. The Journal of Clinical Endocrinology & Metabolism. 2015;100(5):E697-E709. 8. von Roemeling CA, Marlow LA, Wei JJ, Cooper SJ, Caulfield TR, Wu K, Tan WW, Tun HW, Copland JA. Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma. Clinical Cancer Research. 2013 May 1, 2013;19(9):2368-80. 9. Noto A, Raffa S, De Vitis C, Roscilli G, Malpicci D, Coluccia P, Di Napoli A, Ricci A, Giovagnoli MR, Aurisicchio L, Torrisi MR, Ciliberto G, Mancini R. Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells. Cell Death Dis. 2013;4:e947. 10. Ben-David U, Gan Q, Golan-Lev T, Arora P, Yanuka O, Oren Y, Graf M, Garippa R, Boehringe M, Gromo G, Benvenisty N. Selective Elimination of Human Pluripotent Stem Cells by an Oleate Synthesis Inhibitor Discovered in a High-Throughput Screen. Cell Stem Cell. 2013 11. Igal RA. Stearoyl-CoA desaturase-1: a novel key player in the mechanisms of cell proliferation, programmed cell death and transformation to cancer. Carcinogenesis. 2010 September 1, 2010;31(9):1509-15. 12. Falvella FS, Pascale RM, Gariboldi M, Manenti G, De Miglio MR, Simile MM, Dragani TA, Feo F. Stearoyl-CoA desaturase 1 (Scd1) gene overexpression is associated with genetic predisposition to hepatocarcinogenesis in mice and rats. Carcinogenesis. 2002 November 1, 2002;23(11):1933-6. This research was funding in part by grants from the Mayo Center for Biomedical Discovery and Mayo Clinic NCI Designated Cancer Center, Deputy Director Discretionary Fund. SCD1 protein expression is elevated in HCC versus normal liver tissues as shown by (A) immunohistochemistry and (B) quantitative analysis. (C) SCD-1 mRNA in TCGA HCC tumors is elevated compared to adjacent non- tumoral tissues. Stearoyl-CoA desaturase-1: a novel key player in the mechanisms of cell proliferation, programmed cell death and transformation to cancer. Stearoyl-CoA desaturase-1 (SCD1) is the rate limiting enzyme converting saturated to monounsaturated fatty acids. Regulation of MUFA/SFA balance in mammalian cell lipids by SCD1. CE, cholesterol esters; PL, phospholipids; TAG, triacylglycerols. From Igal et al. Carcinogenesis. 2010;31(9):1509-1515. Metabolic and signaling regulations by SCD1 in cancer cells. ACL, adenosine triphosphate-citrate lyase; AMPK, AMP-activated protein kinase; TCA, tricarboxylic acid cycle. From Igal et al. Carcinogenesis. 2010;31(9):1509-1515. Biosynthesis and transfer of unsaturated fatty acids to activate Wnts Biosynthesis of palmitoleic acid. Acetyl-CoA carboxylase catalyzes the transformation of acetyl-CoA to malonyl-CoA. Palmitic acid is generated in a multistep process from malonyl-CoA by fatty acid synthase (FAS) and is then activated by fatty acyl–CoA synthetase. The palmitoyl-CoA thus obtained undergoes unsaturation to palmitoleoyl-CoA by stearoyl-CoA desaturase 1 (SCD1). Porcupine preferentially utilizes the pool of palmitoleoyl-CoA to fatty acylate Wnts at a conserved serine residue (S209 in Wnt3a), thereby forming an ester linkage. Aaron H Nile & Rami N Hannoush Fatty acylation of Wnt proteins Nature Chemical Biology 12, 60–69, 2 015 C. Western analyses showed that survivin and cMyc were elevated in concert with SCD1 expression in cell lines responsive to SSI-4. D. Western analyses demonstrated that SSI-4 induces apoptosis (PARP cleavage & cleaved caspase 3) and ER stress (BIP upregulation), as well as attenuation of Wnt regulated genes (survivin, c-Myc & cyclin D1). Oleic acid co-treatment rescues the SSI-4 effect demonstrating SCD1 specificity. E. HepG2 cells silenced for cMyc (shRNA) led to ~76% attenuation of cell proliferation. Dose out with SSI-4 provided small benefit to further growth inhibition indicative that cMyc is a major driver of proliferation. Fig. 3. SSI-4 inhibits tumor growth in HCC PDX. A. IHC staining of patient matched tumor and PDX tissues showed equivalent SCD1 expression. B. PAX148 tumor tissues (5 mm 3 ) with 50% matrigel were implanted in the right flank of 8 week old female athymic nude mice. At ~100 mm 3 , daily SSI-4 (50 mg/kg oral) was begun. Tumor volume and mouse weight were monitored twice weekly. Tumor volume was calculated using (length x width x height/0.523 = mm 3 ) and is reported as the mean ± S.E. of 10 mice. C. IHC showed that nuclear Ki-67, cMyc and survivin protein are attenuated in response to SSI-4 in mice. ER stress is induced as evidenced by BIP upregulation. H score is calculated based upon signal intensity (0–3) using the formula: [(1+%x1) + (2+%x2) + (3+%x3)]. * indicates statistical significance (P>0.05).
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CONCLUSIONS BACKGROUND
Targeting stearoyl CoA desaturase 1 (SCD1) in hepatocellular carcinoma Ilah Bok1, Laura A. Marlow1, James L. Miller1, Jason Hall1, Akiko Matsuda2, Yan Asmann3, Catherine D. Moser4, Vivekananda Sarangi5,
Steven R. Alberts6, Kabir Mody7, Lewis R. Roberts4, Mark J. Truty8, Tushar C. Patel2, John A. Copland1 1Department of Cancer Biology, 2Gastroenterology and Hepatology Department, 3Health Science Research, 7Hematology Oncology, Mayo Clinic Florida, Jacksonville, Florida;
4Gastroenterology; 5Health Science Research, 6Medical Oncology, 8General and GI Surgery, Mayo Clinic, Rochester, MN
Abstract 192 / 21 April 2, 2017 1 – 5 pm
FUTURE DIRECTIONS
RESULTS
REFERENCES & ACKNOWLEDGEMENTS
Fig. 1. A novel SCD1 inhibitor demonstrated SCD1 specificity and excellent oral bioavailability.
Figure 4: SSI-4 inhibits HCC cell proliferation, demonstrating synergy with sorafenib.
Objectives: Objectives include demonstration antitumor activity of the lead SCD1 inhibitor, SSI-4, against HCC in cell culture and animal models for HCC. HCC is a Wnt driven cancer. SCD1 mechanisms of action are examined that include Wnt regulation and endoplasmic reticulum (ER) stress. SSI-4 was examined for synergy with FDA approved drugs for HCC such as sorafenib.
Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models.
Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing (Data not shown). SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins.
Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC.
Results:
1. We developed novel, high affinity and specific small molecule SCD1 inhibitors through a combined computational and synthetic chemistry approach.
2. SSI-4, the lead SCD1 inhibitor, possesses excellent bio-availibility and can be taken orally with a half-life of 3-4 hours.
3. SSI-4 possesses single agent antitumor activity in HCC PDX mouse models.
4. Soft agar growth predicts SCD1 responsive HCC cell lines.
5. In cell culture, SSI-4 demonstrates anti-proliferative synergy with sorafenib.
Conclusions:
• Targeting fatty acid metabolism may be a novel therapeutic strategy in
treating HCC.
• Identify additional combination therapies that may provide antitumor synergy with SCD1 inhibitors.
• Demonstrate in vivo antitumor synergy with sorafenib and other novel combination therapies.
• Develop our novel lead SCD1 inhibitor, SSI-4, for IND filing and FDA approval towards clinical trials.
Figure 4. SCD1 inhibits HCC cell proliferation and demonstrates synergy with sorafenib. A. Dose response of 0.001 – 10 nM SSI-4 with 5 day exposure demonstrates IC50 of 1-3 nM in HCC responsive cell lines. B. Single cells grown in 3D soft agar identified SCD1 sensitive cell lines. C. Sorafenib dose out with same conditions as SSI-4 showed proliferation inhibition for all HCC cell lines. IC50 values are shown. D. Using the method of Chou and Talalay, combination index (CI) showed synergy with combined SSI-4 and sorafenib in HLF cells.
Figure 2: SCD1 is overexpressed in HCC and linked to Wnt regulation in HCC
Fig. 3. SSI-4 inhibits in vivo tumor growth in HCC patient derived xenograft (PDX) mouse models
The paucity of effective therapeutic agents for hepatocellular carcinoma (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor against HCC.
ABSTRACT
A. Structure of SSI-4 and SSI-2. B. SCD1 inhibitor candidates were evaluated for their inhibition of oleoyl CoA biosynthesis by mouse liver microsomes in vitro. SSI-2 or SSI-4 were incubated with microsomes and 13C18-labeled stearoyl CoA, a native substrate of SCD1. Bioconversion of this substrate to 13C18-labeled oleoyl CoA product was quantified by LC/MS for drug treatments relative to vehicle (DMSO) controls. MF-438, a commercially available SCD1 inhibitor, is used as a comparator. SSI-1 and SSI-3 IC50 values were >1 uM (data not shown). C. HCC cell lines demonstrate inhibition of proliferation with 1 µM SSI-4. To demonstrate SCD1 specificity, cells were pretreated with 5 µM of oleic acid to rescue the growth inhibitory phenotype. Doubling times predict response to SSI-4 inhibition. D. A single dose of oral (PO) or intravenous (iv) SSI-4 was administered to fasted male C57BL/6 mice with plasma levels measured over 24 hours. E. Half-life and bioavailability are key parameters measured in the single dose analyses. Bioavailability (%) was calculated with AUC0-last and nominal dose.
. 1. Nile AH, Hannoush RN. Fatty acylation of Wnt proteins. Nat Chem Biol. 2016;12(2):60-9. 2. Cassidy JW, Caldas C, Bruna A. Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts. Cancer Research. 2015 August 1,
2015;75(15):2963-8. 3. Powers T. Cell Growth Control: mTOR Takes on Fat. Molecular Cell. 2008;31(6):775-6. 4. Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer. 2007;7(10):763-77. 5. Porstmann T, Griffiths B, Chung Y-L, Delpuech O, Griffiths JR, Downward J, Schulze A. PKB//Akt induces transcription of enzymes involved in
cholesterol and fatty acid biosynthesis via activation of SREBP. Oncogene. 2005;24(43):6465-81. 6. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434(7035):843-50. 7. von Roemeling C, Laura A. Marlow L, Pinkerton A, Tun H, Smallridge R, Copland J. Aberrant Lipid Metabolism in Anaplastic Thyroid Carcinoma
Reveals Stearoyl CoA Desaturase 1 as a Novel Therapeutic Target. The Journal of Clinical Endocrinology & Metabolism. 2015;100(5):E697-E709. 8. von Roemeling CA, Marlow LA, Wei JJ, Cooper SJ, Caulfield TR, Wu K, Tan WW, Tun HW, Copland JA. Stearoyl-CoA Desaturase 1 Is a Novel
Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma. Clinical Cancer Research. 2013 May 1, 2013;19(9):2368-80. 9. Noto A, Raffa S, De Vitis C, Roscilli G, Malpicci D, Coluccia P, Di Napoli A, Ricci A, Giovagnoli MR, Aurisicchio L, Torrisi MR, Ciliberto G, Mancini
R. Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells. Cell Death Dis. 2013;4:e947. 10. Ben-David U, Gan Q, Golan-Lev T, Arora P, Yanuka O, Oren Y, Graf M, Garippa R, Boehringe M, Gromo G, Benvenisty N. Selective Elimination of
Human Pluripotent Stem Cells by an Oleate Synthesis Inhibitor Discovered in a High-Throughput Screen. Cell Stem Cell. 2013 11. Igal RA. Stearoyl-CoA desaturase-1: a novel key player in the mechanisms of cell proliferation, programmed cell death and transformation to cancer.
Carcinogenesis. 2010 September 1, 2010;31(9):1509-15. 12. Falvella FS, Pascale RM, Gariboldi M, Manenti G, De Miglio MR, Simile MM, Dragani TA, Feo F. Stearoyl-CoA desaturase 1 (Scd1) gene
overexpression is associated with genetic predisposition to hepatocarcinogenesis in mice and rats. Carcinogenesis. 2002 November 1, 2002;23(11):1933-6.
This research was funding in part by grants from the Mayo Center for Biomedical Discovery and Mayo Clinic NCI Designated Cancer Center, Deputy Director Discretionary Fund.
SCD1 protein expression is elevated in HCC versus normal liver tissues as shown by (A) immunohistochemistry and (B) quantitative analysis. (C) SCD-1 mRNA in TCGA HCC tumors is elevated compared to adjacent non-tumoral tissues.
Stearoyl-CoA desaturase-1: a novel key player in the mechanisms of cell proliferation, programmed cell death and transformation to cancer.
Stearoyl-CoA desaturase-1 (SCD1) is the rate limiting enzyme converting saturated to monounsaturated fatty acids.
Regulation of MUFA/SFA balance in mammalian cell lipids by SCD1. CE, cholesterol esters; PL, phospholipids; TAG, triacylglycerols. From Igal et al. Carcinogenesis. 2010;31(9):1509-1515.
Metabolic and signaling regulations by SCD1 in cancer cells. ACL, adenosine triphosphate-citrate lyase; AMPK, AMP-activated protein kinase; TCA, tricarboxylic acid cycle. From Igal et al. Carcinogenesis. 2010;31(9):1509-1515.
Biosynthesis and transfer of unsaturated fatty acids to activate Wnts
Biosynthesis of palmitoleic acid. Acetyl-CoA carboxylase catalyzes the transformation of acetyl-CoA to malonyl-CoA. Palmitic acid is generated in a multistep process from malonyl-CoA by fatty acid synthase (FAS) and is then activated by fatty acyl–CoA synthetase. The palmitoyl-CoA thus obtained undergoes unsaturation to palmitoleoyl-CoA by stearoyl-CoA desaturase 1 (SCD1). Porcupine preferentially utilizes the pool of palmitoleoyl-CoA to fatty acylate Wnts at a conserved serine residue (S209 in Wnt3a), thereby forming an ester linkage. Aaron H Nile & Rami N Hannoush Fatty acylation of Wnt proteins Nature Chemical Biology 12, 60–69, 2 015
C. Western analyses showed that survivin and cMyc were elevated in concert with SCD1 expression in cell lines responsive to SSI-4. D. Western analyses demonstrated that SSI-4 induces apoptosis (PARP cleavage & cleaved caspase 3) and ER stress (BIP upregulation), as well as attenuation of Wnt regulated genes (survivin, c-Myc & cyclin D1). Oleic acid co-treatment rescues the SSI-4 effect demonstrating SCD1 specificity. E. HepG2 cells silenced for cMyc (shRNA) led to ~76% attenuation of cell proliferation. Dose out with SSI-4 provided small benefit to further growth inhibition indicative that cMyc is a major driver of proliferation.
Fig. 3. SSI-4 inhibits tumor growth in HCC PDX. A. IHC staining of patient matched tumor and PDX tissues showed equivalent SCD1 expression. B. PAX148 tumor tissues (5 mm3) with 50% matrigel were implanted in the right flank of 8 week old female athymic nude mice. At ~100 mm3, daily SSI-4 (50 mg/kg oral) was begun. Tumor volume and mouse weight were monitored twice weekly. Tumor volume was calculated using (length x width x height/0.523 = mm3) and is reported as the mean ± S.E. of 10 mice. C. IHC showed that nuclear Ki-67, cMyc and survivin protein are attenuated in response to SSI-4 in mice. ER stress is induced as evidenced by BIP upregulation. H score is calculated based upon signal intensity (0–3) using the formula: [(1+%x1) + (2+%x2) + (3+%x3)]. * indicates statistical significance (P>0.05).
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