Up Regulation Of Platelet-derived Growth Factor-A Is Responsible For The Failure Of Restarted Interferon α Treatment In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), one of the most common malignanciesworldwide and the second cancer killer in China, is a hypervascular tumor, andabundant and tortuous blood vessels distinguish HCC from benign lesions inangiography. The first clues to the endogenous angiogenesis inhibitors came with theobservations that interferon. (IFN-α) may inhibit endothelial cell chemotaxis andproliferation, and IFN-. has been proven to effectively inhibit HCC growth andrecurrence, especially when combined with cytotoxic drugs. Although severalsignaling pathways are involved in the angiogenesis, attention has been focused onthe vascular endothelial growth factor (VEGF) family, especially VEGF-A. Thecritical involvement of VEGF-A in vascular development and angiogenesis is to bindand activate of VEGF receptor-2, which stimulates endothelial migration andproliferation. IFN-. inhibits tumor cells production of VEGF and endothelial cellmotility, and demostrates direct and indirect anti-angiogenic activity. Nowadays,IFN-. is applied in the treatment of HCC, renal cancer, neuroendocrine tumors,prostate cancer, malignant melanoma, chronic myelogenous leukemia, and superficialbladder carcinoma,We previously reported that IFN-. inhibited tumor growth and recurrence innude mice bearing human HCC xenografts, the same is true of the nude mice withhuman HCC after curative resection, mediated by anti-angiogenic effect throughdown-regulation of VEGF but not basic fibroblast growth factor (bFGF). In theLCI-D20 model, anti-angiogenic activity was a more important factor than theanti-proliferative effect of p48-ISGF, protein deficiency, which plays a pivotal role inthe IFN-αassociated signal transduction pathway. Despite the conspicuous decreasein tumor burden and vasculature induced by the treatment with IFN-., little is knownabout the reversibility of these changes after IFN-. therapy is discontinued.A randomized control trial showed that 18 months' post-operative IFN-.treatment delayed recurrence and prolonged patients' survival. Unfortunately, tumorsrecurred in a number of patients shortly after IFN-αtreatment was discontinued. Thespeed of vascular regrowth in HCC after IFN-. therapy is discontinued is clinicallyrelevant and may influence the use of IFN-.. Based on the results of our clinical trial and pilot study, this experimental design investigates whether relapse is inevitable,and whether restarted IFN-. treatment is effective. The results demonstrate that withvascular regrowth after IFN-. therapy is discontinued, PDGF-A expression increasesdramatically when IFN-αtreatment is restarted. There is no obvious decrease intumor burden and microvessel density (MVD). Pericytes are another vascular celltype that provides endothelial cells with crucial survival signals, and PDGF/PDGFRpathway regulated pericyte homeostasis, so we postulate that up-regulation ofPDGF-A is responsible for the failure of restarted IFN-αtreatment for HCC. To testthis hypothesis, we added imatinib (Gleevec, Novartis, Basel, Switzerland), thePDGF-receptor inhibitor, to the restarted IFN-αtreatment course, and resulted insignificant inhibition of tumor growth and angiogenesis. Overall, the results indicatethat the up-regulation of PDGF-A was responsible for the failure in the restartedIFN-αtreatment when tumors recurred after the first IFN-αtherapy was discontinued,and long-term disease control can be best achieved by combination therapy withimatinib in an IFN-αrestarted treatment course.1. Restarted IFN-αtreatment was less effective in suppressing tumor growth than the first treatment courseWhen IFN-αtreatment group was compared with the control group, a significantdecrease of tumor weight was observed (0.27±0.19 g versus 0.68±0.24g), MVD wasalso markedly decreased [22/HP(95% CI=15-29) versus 46/HP (95% CI=32-60)].Furthermore, ELISA results showed that the serum VEGF levels were lower in theIFN-αtreatment group than in control group A (24.5±8.7 pg/ml versus 41.6±12.0pg/ml), P＜0.05. When IFN-αtreatment was discontinued for 20 days, there was nosignificant difference in the tumor weight between the follow -up of control group andfollow-up of IFN-αtreatment group. Furthermore, the restarted IFN-αtreatment didnot result in significant inhibitory effect on tumor growth and MVD when comparewith control group [restrated group F: 3.22±0.62 g, 61/HP (95% CI=48-74), controlgroup E: 3.81±0.92 g, 70/HP (95% CI=59-81)], P＞0.05.2. Response of IFN-αtreatment was not influenced by pretreatment tumor weight.The poorer tumor response to the restarted treatment of IFN-αmay have resultedfrom a larger tumor load. To test this possibility, tumors were allowed to grow for 35days when the tumor weight was about 1.5 g (the same size as the tumor before the restarted IFN-αtreatment). IFN-αtreatment for 20 days resulted in a significantreduction of tumor weight compared with control group G, being 2.28±0.63 g versus3.90±0.80 g (P=0.003).3. Changes in the angiogenesis-related gene expression profile of human HCC in nude mice treated by IFN-α.In two independent cDNA microarray analyses, the results showed that IFN-αtreatment down-regulated several pro-angiogenic factors, including VEGF,angiogenin, bFGF, PDGF-A, TGF, TNF, G-CSF, EGF, IGF, and IL-8 in the firsttreatment course. All the pro-angiogenic factors except PDGF-A were againdown-regulated in restarted IFN-αtreatment group compared with control group.4. Effects of IFN-αon the expression of VEGF165, PDGF-A and VEGF receptor 2.RT-PCR with primers showed two transcripts whose base pair lengthcorresponded to VEGF165 and VEGF121, and a minor transcript corresponding toVEGF189. However, Western blotting results showed no immunoreactive proteinsother than VEGF165. These data indicated that IFN-αdown-regulated the expressionof VEGF165.To verify the results from the cDNA microarray analyses, we used RT-PCR andWestern blot assays to study the expression of VEGF165 and PDGF-A. The results ofRT-PCR showed that IFN-αdecreased the VEGF165 expression in the first (IFN-αtreatment group) and restarted IFN-αtreatment course (restarted IFN-αtreatmentgroup) compared with their co ntrol groups, respectively, which corresponds with thechange in serum VEGF concentration.Western blot assays for VEGF165 of HCC tissues under reducing conditionsshowed two bands of 23 and 21 kD, respectively, which is consistent with twoglycosylation variants of VEGF165, as reported previously. No other isoforms ofVEGF were detected. Tumors from IFN-αtreatment group expressed a lower level ofVEGF165 than those of control group in the first treatment course. Similarly, in thesecond treatment course, the VEGF165 concentration of restarted IFN-αtreatmentgroup was lower than that of restarted control group. Thus, VEGF165 expression inthe first and second treatment courses was down-regulated by IFN-αtreatment.The results of RT-PCR show that PDGF-A expression diminished in tumorstreated with IFN-αcompared with the control group in the first treatment course, andincreased in the restarted IFN-αtreatment group compared with its control group. These results are consistent with the cDNA microarray analysis. Western blot assaysshowed that PDGF-A in IFN-αtreatment group was down-regulated significantlycompared with its control group. In contrast, restarted IFN-αtreatment group had ahigher PDGF-A expression level than its control group in the second treatment courseTo assess the inhibiting effect of IFN-αon the VEGFR2 signaling, thephosphorylation status of VEGFR2 was determined using antibodies that recognize aphosphorylation site in the VEGFR2 kinase domain of tyrosine 951 (Y951), whichplays a critical role in pathological angiogenesis. Western blot assays results showedthat VEGFR2 phosphorylation of Y951 was detected at a high level in control groups,but at a very low level in IFN-αtreatment group and restarted IFN-αtreatment group.Thus, both the VEGF165 level and VEGFR2 phosphorylation demonstrated that theIFN-αretains its ability to inhibit VEGF signaling in the first and second treatmentcourses.5. Blockage of PDGF signaling by imatinib significantly improved the efficacy of restarted IFN-αtreatment.Combination treatment with imatinib (PDGF receptor inhibitor) and IFN-αresulted in a significant decrease in tumor burden in the second treatment coursecompared to IFN-αtreatment alone (P=0.01) and imatinib treatment alone (P=0.018), which was accompanied by a decrease in angiogenesis estimated by MVD(P=0.007 for IFN-αtreatment alone; P=0.004 for imatinib treatment alone).6. The high expression of transcription factor HIF-2αin the restarted IFN-αtreatment may trigger the high expression of PDGF-A.The HeLa and MHCC07-H,MHCC07-L cells exposed to hypoxic conditionsexpressed high level of hypoxia inducible factor-1αand 2αcompared with cells in thenormoxic conditions. HIF-1αand HIF-2αare both undergo rapid hypoxia-inducedprotein stabilization and bind identical target DNA sequences. In the first IFN-αtreatment course, the expression of HIF-1αand HIF-2αin the IFN-αtreatment groupB is lower than in the control group A; in the restarted IFN-αtreatment group F, theexpression of HIF-1αwas down regulated again compared with control group E,while HIF-2αmaintained high level in the restarted IFN-αtreatment group F.7. Correlation of contrast agent flow and immunohistochemical results.Contrast-enhanced ultrasonography were performed at day 9, 12, 17, 27, and 42.For the entire cohort (treated and control tumors), AUC and AT were positively andnegatively correlated with MVD and the tumor volume and weight. Conclusions1. Restarted IFN-αtreatment was less effective in suppressing tumor growththan the first treatment course by evasion of antiangiogenic targeting of VEGFsignaling.2. Response of IFN-αtreatment was not influenced by pretreatment tumorweight.3. Up regulation of platelet -derived growth factor-Ais responsible for the failureof restarted interferonαtreatment in hepatocellular carcinoma, and blockage ofPDGF signaling by imatinib significantly improved the efficacy of restarted IFN-αtreatment.4. Quantification of intratumor flow of ultrasonographic contrast agent atgray-scele imaging shows promise for monitoring tumor vascular response toanti-angiogenic therapy.The potential application of this work1. The differential expressed genes, in the first and restrated IFN-αtreatment,identified by angiogenesis associated cDNA microarray analysis provide a valuableresource for basic and clinical studies of angiogenesis in the study of HCC metastasis.2. Restarted IFN-αtreatment was not effective because of up-regulation ofPDGF-A and the VEGF signal pathway remained inhibited. The combination ofPDGF-A inhibitor improved the restarted IFN-αtreatment, and may be a tractableclinical strategy for treating recurrent HCC.3. Quantification of intratumoral flow of ultrasonographic contrast agent atgray-scale imaging shows promise for monitoring tumor vascular response toanti-angiogenic therapy.Originalities of this work1. Analysed and confirmed, for the first time, that restarted IFN-αtreatment isnot effective in inhibiting HCC growth because of up-regulation of PDGF-A, and thecombination of PDGF receptor inhibitor imatinib improved the restarted IFN-αtreatment.2. Established the platform of using contrast-enhanced ultrasonography toassess the response of anti-angiogenic agents on the HCC in the LCI-D20 model, andfound the AUC and AT were positively and negatively correlated with current histologic indices for quantifying angiogenesis MVD.