Radiofrequency ablation (RFA) is a potentially curative therapy for hepatocellular carcinoma (HCC). However, incomplete RFA can induce accelerated invasive growth at the periphery. The mechanisms underlying the RFA-induced tumor promotion remain largely unexplored. Three human HCC cell lines were exposed to 45°C-55°C for 10 minutes, simulating the marginal zone of RFA treatment. At 5-12 days post-treatment cell proliferation, parameters of epithelial-mesenchymal transition (EMT), and activation of mitogen-activated protein kinases were analyzed. Livers from patients with viral hepatitis without and with HCC (n = 114) were examined to confirm the relevance of altered kinase patterns. In vivo tumorigenic potential of heat-treated versus untreated HCC cells was studied in nude mice. Heating to 55°C killed all HCC cells, whereas 65%-85% of cells survived 48°C-50°C, developing spindle-like morphology and expressing CD133, cytokeratin (CK)7, CK19, procollagen-α1(I), and Snail at day 5 after heat exposure, which returned to baseline at day 12. Heat-exposed HCC cells showed enhanced proliferation and prominent activation of p46-Shc (Src homology and collagen) and downstream extracellular signal-related kinase (Erk)1/2. In patients, Shc expression correlated with malignant potential and overall survival. Blocking Erk1/2 reduced proliferation and EMT-like changes of heat-treated HCC cells. Implantation of heat-exposed HEPG2 cells into nude mice induced significantly larger, more aggressive tumors than untreated cells. Conclusions: Sublethal heat treatment skews HCC cells toward EMT and transforms them to a progenitor-like, highly proliferative cellular phenotype in vitro and in vivo, which is driven significantly by p46Shc-Erk1/2. Suboptimal RFA accelerates HCC growth and spread by transiently inducing an EMT-like, more aggressive cellular phenotype. (Hepatology 2013;58:1667–1680)
Radiofrequency ablation (RFA) is accepted as a potentially curative therapy for the early stages of primary hepatocellular carcinoma (HCC). RFA induces tumor necrosis with low complication rates and is superior to percutaneous ethanol injection in tumor ablation. However, suboptimal RFA treatment for HCC has been reported as a risk factor of early diffuse recurrence. Large tumor size is a major risk factor of local recurrence because of poorly defined margins. Such recurrent HCC appears to behave more aggressively than before RFA[5-8] and significantly reduces overall survival (OS) of HCC patients. Phenotypic and functional alterations of HCC cells subjected to heat treatment have not been studied.
Epithelial-mesenchymal transition (EMT) is thought to be a critical factor in progression of cancer and dictating metastasis. Several oncogenic pathways (such as those of growth and transcription factors, integrins, Wnt/β-catenin, and Notch) can induce EMT. In particular, the Ras/extracellular signal-related kinase (Erk)1/2 pathway has been shown to activate two EMT-related transcription factors, namely, Snail and Slug. A recent clinical study has shown a correlation of Snail transcript levels with capsular and portal invasion of HCC. Other inducers of EMT in HCC are TWIST1 and CHD1L.[13, 14] Moreover, expression of type I procollagen (COL1A1) is a useful marker of transition to a mesenchymal phenotype.
Src homology and collagen (Shc) is a central SH2-containing cytoplasmic adaptor protein, which directly binds to tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor beta (PDGFRβ), insulin-like growth factor 1 receptor (IGF-1R), and fibroblast growth factor receptor (FGFR). Notably, Shc is a central player in malignant transformation.[16-20] Mitogenic, transforming, and proinvasive signal transduction from Shc to mitogen-activated protein kinases (MAPKs) by Grb2-Sos has been well studied,[21-23] with p46-Shc and p52-Shc being central upstream regulators of MAPK activation, whereas the alternatively spliced p66-Shc isoform appears to promote apoptosis.[24, 25] However, the role of Shc isoforms in liver diseases is still poorly understood. Although we previously reported that p46-Shc phosphorylation is a hallmark of hepatocarcinogenesis and liver regeneration in rats,[26, 27] the role of Shc in human HCC has not been studied yet.
Here, we demonstrate that sublethal heat treatment of HCC cells, as might occur in marginal zones of RFA therapy, endows these cells with a higher proliferative and carcinogenic potential in vitro and in vivo. These properties are linked to EMT-like changes and appear driven by p46-Shc and Erk1/2 activation.
Local recurrences of HCC can progress rapidly after RFA,[5, 6], and cancer cells up-regulate CK19 (i.e., a feature of cholangiocarcinoma and hepatic progenitor cells).[34, 35] Recent studies also describe other progenitor cell biomarkers, such as CD133, that characterize HCC with enhanced malignant potential.[36, 37] Here, we demonstrate that hepatoma cells that were exposed to sublethal heat for 10 minutes adopted molecular and functional characteristics of hepatic progenitors (CK7, CK19, and CD133), coupled with increased proliferation, up-regulation of genes that are involved in EMT (TWIST1, Snail, COL1A1, and CHDL1) and an enhanced malignant potential in vivo. Moreover, the observed EMT and aggressiveness of HCC cells exposed to sublethal heat were dependent on activation of the MAPKs, Erk1/2 (and upstream Shc).
We describe and analyze, for the first time, that heat-exposed HCC cells undergo EMT-like changes, gain progenitor characteristics, and display a higher malignant potential. We note that activation of Erk1/2 has recently been identified as an important regulator of EMT in tight association with Snail.[38, 39] Three types of EMT are known: Type 1 describes the invasion of transitional cells into the mesenchyme during development; type 2 occurs when epithelia transform into myofibroblast-like cells during wound healing and repair; and type 3 is the adoption of mesenchymal properties by cancer cells that permit their infiltration and migration into the circulation to generate distant metastases. Thus, Snail and TWIST1 can induce type 3 EMT in pancreatic and breast cancer cells,[41, 42] and TWIST1 protein has also been shown to directly trigger type 3 EMT and promote invasion by activation of Ras. Taken together, our data indicate that sublethal heat exposure of HCC promotes type 3 EMT by induction of Snail, TWIST1, and other (functional) EMT markers and upstream p46Shc-Erk1/2 activation.
Another novel finding of our study is the likely important upstream role of Shc in HCC progression in general and after sublethal heat treatment. p46-Shc and its phosphorylation are clearly enhanced after heat exposure, which is upstream of p-Erk1/2 activation, whereas p-SAPK/JNK and p38 MAPK remained unchanged. Shc functions as an adapter molecule of the EGFR and other tyrosine kinase receptors, such as PDGFRβ, IGF-1R, and FGFR, involved in oncogenic activation.[16-20] The signaling cascade induced by Shc activation, named the alternative pathway, is thought to be a master regulator of tumor growth, differentiation, and development.[17, 45] p-Erk1/2 itself is a well-known regulator of cell proliferation, malignant transformation, and tumor progression. Enhanced expression of Shc, especially activated p46-Shc, is a general phenomenon in hepatocarcinogenesis. In this line, we showed that Shc expression strongly correlated with a serum marker of enhanced malignant potential (AFP-L3) and overall patient survival. Heat treatment (50°C) activated p46-Shc in HEPG2 cells and activated p-Erk1/2. Thus, these data suggest that p46-Shc expression, and its activation by phosphorylation, is a central switch for activation of Erk1/2, which then accelerates both malignant transformation and tumor progression in HCC after sublethal heat exposure.
In our previous study of spontaneous hepatocarcinogenesis in the Long-Evans Cinnamon rat, we showed that total activated Shc (p46- and p52-Shc) was highly increased in hepatoma cells, with a prominent activation of p46-Shc in HCC specimens. We could also demonstrate that p46-Shc was strongly up-regulated in the early stage of liver regeneration in rats with 70% hepatectomy, supporting its role also as a primary inducer of hepatocyte regeneration. At present, it is unclear why only phosphorylated p46Shc is up-regulated during proliferation.
A recent report that employed the hepatic implantation model of VX2 carcinoma (an anaplastic squamous cell carcinoma in rabbits) supports our findings, although conditions were less well defined and the data largely descriptive. The researchers carried out RFA with three temperature settings (55°C, 70°C, and 85°C) for 5 minutes. Only the 55°C treatment significantly induced a more rapid progression of remnant tumor, with increased protein levels of proliferating cell nuclear antigen, matrix metalloproteinase 9, vascular endothelial growth factor (VEGF), hepatocyte growth factor, and interleukin-6 in tumor tissue. Another report showed that an aggressive and heat-resistant subclone of HepG2 cells expressed higher levels of hypoxia-inducible factor 1 alpha and VEGF-A in vitro and in nude mice in vivo, suggesting that heat treatment may also promote tumor angiogenesis.
HSPs are known inhibitors of apoptotic cell death and inducers of proliferation and invasion/metastasis in gastrointestinal cancer cells. We demonstrated that heat treatment at 48°C and 50°C significantly induced HSP27, HSP70, and HSP90 proteins at day 5. The elevated expression of these HSPs very likely contributes to the higher malignant potential of the heat-treated hepatoma cells, as was previously reported.
Notably, we demonstrated that inhibition of Erk1/2, which is downstream of Shc, almost normalized Ki-67, CyclinD1, Snail, COL1A1, CK19, and CD133 expression and almost completely reverted the EMT- and progenitor-like phenotype of heat-exposed HCC cells.
Importantly, we could verify our in vitro data by implanting HEPG2 cells into nude mice with or without previous heat treatment. Here, cells with previous exposure to 48°C/50°C induced an impressive, higher in vivo tumor growth, which was accompanied by enhanced proliferation and up-regulation of some, but not all, markers of EMT.
The finding of only modest EMT-like changes in vivo at the day of tumor harvest (18 days after implantation of cells) can be explained by the kinetics of the observed EMT in vitro, because all the changes found in heat-exposed HCC cells at day 5 returned to baseline at day 12. Our data also indicate that the heat-induced EMT-like changes with activation of p46-Shc and Erk1/2 in HCC are reversible in vivo as well, and that invasion and metastasis of HCC may also transiently occur within a short time frame after RFA. The reversibility of the EMT phenotype is important because it largely rules out that heat treatment generates aggressive subclones, and thus potential artifacts, but rather shows that this mechanism is operative and of relevance for in vivo HCC in general. This knowledge may provide a rational basis for the short-term use of adjuvant antiproliferative therapy in RFA-treated HCC. Moreover, we show that heat exposure affects HCC cell lines to a different degree. Because this will likely apply to subclones of HCC cells in vivo, observation of single hepatoma cell clones may not be representative of overall tumor response.
In conclusion, although RFA is currently an important, potentially curative therapy for HCC, inadequate or sublethal treatment might induce a higher malignant potential and cause recurrent HCC with a worse prognosis. Our data underline the need to apply sufficiently high temperatures and secure wide therapeutic margins, in an attempt to completely eradicate all residual HCC within the treated focal lesion. Our results further suggest that p46-Shc expression and its phosphorylation may be a strong predictor of malignant transformation, tumor invasion, and metastasis of HCC, and that its downstream effecter Erk1/2 is key to EMT-like changes that confer an enhanced malignant potential in insufficiently heat-treated HCC cells. Finally, Erk1/2 (or further upstream molecules) may be an attractive therapeutic target for a short-term adjuvant therapy to prevent HCC recurrence after RFA therapy.