Growth hormone receptor antagonism downregulates ATP-binding cassette transporters contributing to improved drug efficacy against melanoma and hepatocarcinoma in vivo

Abstract

Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Keywords: ABC transporters; GHA; HCC; drug resistance; growth hormone (GH); growth hormone receptor antagonist (GHRA); melanoma.

Figure 1

GHRA suppresses syngeneic mouse melanoma growth in vivo. (A) Mouse Fluc-B16-F10 cells grafted intradermally on the right flank of syngeneic C57BL6/J wild-type (WT) and GHA male mice (transgenic for bGH G119K GHR antagonist) (n=6). Tumor growth over 4-weeks was followed by luminescent imaging following luciferin injections. Luciferin signal was quantified and plotted on the right. The changes in tumor volume (Fluc-B16-F10 in WT and GHA mice) from digital caliper measurement (B) and tumor mass (C) corroborate the suppressed tumor growth in GHA mice which has markedly lower serum IGF1 levels (D) due to presence of a circulating GHRA. Western-blot analysis of the GH downstream signaling mediators – phosphorylated STAT5, AKT and SRC kinase in the tumors of GHA and WT mice (E). B16-F10 cells in culture when treated for 72-hours with serum collected from WT and GHA mice showed suppressed growth rate in the GHA mouse serum (F). (*p < 0.05, mouse studies – repeated measure using SPSS; cell viability - Students t test, n = 3).

Figure 2

Effect of GHRA on response of syngeneic mouse melanoma tumors to cisplatin treatment in vivo. (A) Mouse Fluc-B16-F10 cells grafted intradermally on the right flank of syngeneic C57BL6/J wild-type (WT) and GHA mice (transgenic for bGH G119K GHR antagonist) (n=8). The changes in tumor volume (Fluc-B16-F10 in WT and GHA mice) from digital caliper measurement and representative tumors post-dissection (A) and tumor mass (B) corroborate suppressed tumor growth in GHA mice and improved tumoral response to cisplatin in the GHA mice. The qPCR analysis of ABC transporter RNA expression involved in multi-drug efflux from the tumors in WT and GHA mice (C) and western-blot assessment of GH downstream signaling and ABC transporter protein levels (D) are shown. (E) The changes in ABC transporter RNA level in B16-F10 cells in culture when treated with serum collected from WT and GHA mice (*p < 0.05, mouse studies – repeated measure using SPSS; other assays - Students t test, n = 3).

Figure 3

Effect of absence of GH on response of syngeneic mouse melanoma tumors to cisplatin treatment in vivo. (A) Mouse Fluc-B16-F10 cells were grafted intradermally on the right flank of syngeneic C57BL6/J wild-type (WT) and GH knockout (GHKO) mice (n=6). The changes in tumor volume (Fluc-B16-F10 in WT and GHKO mice) from digital caliper measurement and representative tumors post-dissection (A) and tumor mass (B) corroborate suppressed tumor growth in GHKO mice but not improved tumoral response to cisplatin in the GHKO mice. Western-blot assessment of GH downstream signaling and ABC transporter protein levels (C) and qPCR analysis of ABC transporter RNA expression involved in multi-drug efflux from the tumors in WT and GHKO mice (D) are shown. (*p < 0.05, mouse studies – repeated measure using SPSS; other assays - Students t test, n = 3). (E) Spearman correlation analysis for transcript levels of GHR and ABC transporter and IGF1R and ABC transporters in 471 human melanoma patients in the TCGA cohort (generated using Linkedomics).

Figure 4

GH signaling drives drug resistance in human HCC cells. (A) Treatment with exogenous GH (50ng/mL) causes activation of STATs 3 and 5, SRC family kinase, ERK1/2 and PI3K-AKT signaling in human HCC cells - Hep-G2 (B) and SK-Hep-1 (C) cells in culture. (D) Doxorubicin or sorafenib tosylate (ST) and/or GH treatment increases ABCB1, ABCC1 and ABCG2 protein expression in human HCC cells – Hep-G2 (E) and SK-Hep-1 (F) in culture. (G, H) Recombinant human GH treatment (at 50 or 500ng/mL) increases drug efflux rate in human HCC cells. (I–L) Recombinant hGH (at 50 or 250ng/mL) suppresses doxorubicin induced growth inhibition in SK-Hep-1 (I) and Hep-G2 cells (J) and sorafenib induced growth inhibition in SK-Hep1 (K) and Hep-G2 cells (L). (*p < 0.05, Students t test, n = 3).

Figure 5

Effect of GHRA on response of syngeneic mouse hepatocellular carcinoma tumors to sorafenib treatment in vivo. (A) Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells respond to bovine GH stimulation in culture showing increased cell viability and STAT5 activation after 72 hours (A) as well as increase in the EC50 dose of sorafenib (B). Additionally, GH treatment increased ABC transporter levels in the cultured Hepa1-6 cells (C). Mouse Hepa1-6 cells grafted subcutaneously on the right flank of syngeneic C57BL6/J wild-type (WT) and GHA mice (transgenic for bGH G119K GHR antagonist) (n=6). The changes in tumor volume (Hepa1-6 in WT and GHA mice) from digital caliper measurement (D) and post-dissection tumor mass (E) show suppressed tumor growth and improved tumoral response to sorafenib in the GHA mice. (F) Spearman correlation analysis for transcript levels of GHR and ABC transporter and IGF1R and ABC transporters in the tumor of 371 HCC patients in the TCGA cohort. (G) Serum IGF1 levels of Hepa1-6 tumor-bearing GHA mice are significantly lower than that of tumor-bearing WT mice. (H–J) Correlation of overall survival probability of 371 HCC patients in the TCGA cohort with RNA expression of IGF1R (H), or ABCB1 (I), or ABCC1 (J). (*p < 0.05, ***p < 0.001, mouse studies – repeated measure using SPSS; other assays - Students t test, n = 3).