Alternative mammalian target of rapamycin (mTOR) signal activation in sorafenib-resistant hepatocellular carcinoma cells revealed by array-based pathway profiling

Abstract

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.

Fig. 1.

Phosphoprofiling of key signaling molecules by RPPA. A, phosphorylation status of 180 signaling nodes in a panel of 95 cancer cell lines cultured in the presence of 10% FCS. Red and blue colors indicate high- and low-level phosphorylation, respectively. STAT, signal transducers and activators of transcription; SAPK/JNK, stress-activated protein kinase/c-Jun NH₂-terminal kinase; RTK, receptor tyrosine kinase; PI3K, phosphatidylinositol 3′-kinase; MAPK, mitogen-activated protein kinase; NFκB, nuclear factor-kappaB; OS, osteosarcoma; OSCC, oral squamous cell carcinoma. B, immunoblot (left) and RPPA (right) analyses of A431 cells cultured without (−) and with (+) EGF for 10 min with anti-p-ERK1/2 (T202/Y204) antibody. The mean fluorescence intensity in arbitrary units (top) and images (bottom) of quadruplicate RPPA spots of lysate undiluted (1) and diluted 1:2 (1/2), 1:4 (1/4), and 1:8 (1/8) -fold are shown (right). C, D, relative p-RPS6 S235/236 (C) and p-Met T1234/1235 (D) expression of 95 cell lines determined via RPPA (right). Cell lines with the three highest and three lowest levels of expression were selected and subjected to immunoblotting with the same antibody (left). Ave, average.

Fig. 2.

p-RPS6 S235/236 correlates with the sensitivity of HCC to sorafenib. A, upper graph, IC₅₀ values for sorafenib against 23 HCC cell lines sorted from the most sensitive (left) to resistant (right) ones. Columns and error bars represent the mean and S.D. of three independent experiments, respectively. Lower panels, immunoblot analysis of pRPS6 S235/236, pRPS6 S240/244, RPS6, and γ-tubulin (loading control) expression in the 23 HCC cell lines. B, correlation between RPPA and immunoblot analyses of p-RPS6 S235/236 expression in the 23 HCC cell lines (R² = 0.733). C, detection of p-RPS6 S235/236 in pretreatment biopsy samples. Hematoxylin and eosin (H&E) (a-d1) and immunoperoxidase staining with anti-p-RPS6 S235/236 (a-d2) and total RPS6 (a-d3) antibodies of HCC biopsy specimens obtained from a responder (patient 1 (a and b)) and a representative non-responder (patient 2 (c and d)) to sorafenib. Original magnification was ×40 (a1–3 and c1–3) and ×200 (b1–3 and d1–3).

Fig. 3.

Alternative mTOR signal activation in sorafenib-resistant HCC cells. A, C, representative sorafenib-sensitive (HUH-6 and HuH-7) and -resistant (JHH-1, JHH-2, SNU-182, SNU423, and SNU-387) HCC cells were treated with the indicated concentrations of sorafenib for 3 h, and the expression of p-RPS6 S235/236, total RPS6, p-ERK1/2 T202/Y204, total ERK, p-RSK T380, total RSK, p-S6K T389, and total S6K was determined via immunoblotting. B, schematic representation of the mTOR and MAPK pathways and their inhibitors. D, representative sorafenib-resistant (JHH-1, JHH-2, SNU-182, SNU423, and SNU-387) HCC cells were treated with the indicated concentrations of everolimus for 3 h, and the expression of p-RPS6 S235/236 and total RPS6 was determined via immunoblotting. E, distribution of IC₅₀ values of representative sorafenib-sensitive (SNU-449, HUH-6, JHH-7, HuH-7, and SK-Hep1) and -resistant (JHH-1, JHH-2, SNU-182, SNU-423, and SNU-387) HCC cells to AZ8055. Boxes indicate 25th to 75th percentiles.

Fig. 4.

Synergy of mTOR and MAPK inhibitors. A, sorafenib-resistant JHH-1 and SNU-182 cells were treated with the indicated concentrations of sorafenib and everolimus, and the expression of p-RPS6 S235/236 and total RPS6 was examined via immunoblotting (top). The bottom panel indicates intensity relative to control blots (no treatment). B, sorafenib-resistant JHH2, SNU-423 and SNU-387 cells were treated with the indicated concentrations of CI-1040, SL0101, and everolimus, and the expression of p-RPS6 S235/236 and RPS6 was examined via immunoblotting (top). The bottom panel indicates blot intensities relative to control blots (no drug treatment). C, sorafenib-resistant SNU-423 cells were treated with the indicated concentrations of CI-1040, and relative cell viability was determined 72 h later. Note that CI-1040 had no significant inhibitory effect on cell growth at 0.1 μm (indicated by a red arrow). D, sorafenib-resistant SNU-423 cells were treated with the indicated concentrations of AZD8055 in the presence (open circles) or absence (solid circles) of 0.1 μm CI-1040, and relative cell viability was determined 72 h later.