Mechanisms of portal vein tumour thrombus formation and development in patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Portal vein tumour thrombus (PVTT) is considered one of most fearful complications of HCC and is strongly associated with a poor prognosis. Clarification of the mechanisms underlying the formation and development of PVTT is crucial for developing novel therapeutic strategies for HCC patients. Several studies have been made to uncover that tumour microenvironment, stem cells, abnormal gene expression and non‐coding RNAs deregulation are associated with PVTT in patients with HCC in the last decade. However, the exact molecular mechanisms of PVTT in patients with HCC are still largely unknown. In the present review, we briefly summarized the molecular mechanisms underlying the formation and development of PVTT in HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is one of the most common and fatal malignancies worldwide. 1 Despite advances in the diagnostic and treatment strategies for different HCC stages, the five-year survival remains extremely low. 2 The presence of portal vein tumour thrombus (PVTT), which is not uncommon, has been identified as one of the most significant factors for poor prognosis in HCC. If left treatment, the median survival time for patients with PVTT is 2.7-4 months, compared with 10-24 months for patients without PVTT. 3,4 The great efforts have been performed to prolong the survival time of HCC patients with PVTT in the past several decades.
Unfortunately, the survival benefit of the various treatment strategies is still extremely limited. [5][6][7] Molecular-targeted cancer therapy, which is ascribed to the increasing understanding of molecular mechanisms for cancer initiation and progression, is considered as a breakthrough for cancer therapy. However, sorafenib and lenvatinib, as molecular-targeted agents recommended for the advanced HCC, are only able to slightly prolong overall survival compared to placebo in HCC patients with PVTT. [8][9][10][11][12][13] Therefore, clarification of the mechanisms underlying the formation and development of PVTT is crucial for developing novel therapeutic strategies for HCC patients.

Multiplemechanismsunderlyingtheformationanddevelopment
of PVTT in HCC have been reported recently. It is well-accepted that PVTT could originate from HCC primary nodules by metastasis, and evidence from several studies showed that gene expression profiles of PVTT tissues (PVTTs) were nearly identical to the corresponding primary HCC tissues (HCCs). [14][15][16][17][18] However, previous studies also showed different gene profiles between PVTTs and HCCs, suggesting that PVTTs do not always have clonal origin from their paired HCCs. Furthermore, tumour microenvironment (including hepatitis B virus (HBV)-associated microenvironment, hypoxia, and extracellular matrix (ECM)), cancer stem cells and non-coding RNAs have been found to contribute to PVTT development. [19][20][21][22][23][24][25] It is noteworthy that the mechanism of PVTT formation and development is far more complicated than previously thought and further great efforts need to be performed. Thus, in this review, we summarize the current state of knowledge and highlight the mechanisms underlying the formation and development of PVTT ( Figure 1 and Table 1).

| ORI G IN S OF PV T T
PVTTcommonlyoccursinpatientswithHCC. 26 whileupto44.0%-62.2%atautopsy. 27 Considering such a relatively high prevalence of PVTT among HCC patients, it has been usually considered as a special type of HCC intrahepatic metastasis in the past several decades. Evidence from several studies also identified PVTT originating from HCC primary nodules by metastasis. Direct evidence from our previous study showed that a PVTT-originated HCCcellline(CSQT-2),establishedbyourgroup,wasabletogenerate PVTT in nude mice after orthotropic inoculation, indicating that PVTT could be formed by HCC cells disseminating from the primary tumour site. 28 Genomic analysis of the paired tumour tissues from HCC patients would uncover whether PVTT originate from the primary HCC tissues. Ye et al. 14  identical gene expression between PVTTs and corresponding HCCs, andfivepatientsacquiredevidentialprogressivealterationsofgene expressions (more than 1000 differentially expressed genes were identified in each patient). 15 Basedontheresults,Wangetal.speculated that most patients had few differentially expressed genes, indicating that PVTTs may be formed by the accumulation of randomly fallen cancer cells from primary HCC tissues. They also considered that PVTTs might be formed by highly invasive sub-clones or therandomlyfallencancercellsacquiredadaptivechangesforthe portal vein microenvironment in those patients with evidential progressive alterations of gene expressions. Indeed, tumour microenvironments, especially hypoxia microenvironment, played a crucial role in regulating genes expression in tumour patients. 30 However, the number of evidential progressive alterations was larger than that of tumour microenvironment-related genes reported previously. 31,32 Hence, this possibility that PVTTs may have different clonal origins from their corresponding HCCs in those patients with evidential progressivealterationscannotberuledout.Moreover,inaprevious study from our group, the clonal relationship of PVTTs with corresponding HCCs was analysed using datasets deposited in a public database.Wefoundthatoneoutof19PVTTswasidentifiedtohave an independent clonal origin from its corresponding HCCs, and the PVTTs with independent clonal origin showed different gene expression and enrichment in biological processes from the primary HCCs. 33 Furthermore, a distinct PVTT (dPVTT), distant from or without liver parenchyma tumour nodules, has been observed in some patients. [34][35][36] To identify the biological features of dPVTT in HCC patients, we collected the matched tumour tissues from 5 HCC patients accompanied by dPVTT in our library, and two-dimensional electrophoresis has been performed to compare the proteome of dPVTT tissues with that of corresponding HCCs. The differentially expressed proteins were identified in dPVTT tissues and HCCs, and protein expression levels with differences of more than threefold were found in at least 80% of the patients. Importantly, we demonstrated that dPVTT showed a more malignant phenotype by detecting the expression of C-Kit. 34 This finding further indicated that some PVTTs did not have the same origin as the paired tumours.
On a more serious note, several studies also identified PVTTs from different HCC patients showed very different gene expression profiles, 15,17,33 implying that the mechanism of PVTT formation is far more complicated than previously thought, and great efforts need to be performed to clarify these inferences further.

| HBV INFEC TI ON AND PV T T
AmountainofevidencehasshownthatHBVinfectionwasstrongly associated with HCC formation and development, and more than 50%ofHCCcasesmaybecausedbypersistentHBVinfection. 37,38 The potential correlation between HBV infection and the formation and development of PVTT also has been investigated for HCC patients in the last decades. In a nationwide study involving 11,950 patients with HCC, the clinicopathological features were compared among HBV-related HCC, hepatitis C virus (HCV)-related HCC and Non-B Non-C HCC patients. The results showed that the incidence ofPVTTinHBV-relatedHCCpatients(31%)washigherthanthatin HCV-related HCC patients (22%) and Non-B Non-C HCC patients TA B L E 1 MechanismintheformationanddevelopmentofPVTTinpatientswithHCC.

| HYP OXIA AND PV T T
Hypoxia, one of the hallmarks of tumours, is common in tumours including HCC. Hypoxia can potentially regulate every aspect of cellular function including growth, proliferation, apoptosis, metastasis, immunity, metabolic reprogramming, self-renewal and others. 51,52 Hypoxia is associated with resistance to chemotherapy and radiotherapy, and is closely related to poor prognosis of HCC. In HCC, hypoxia results from a shortage of blood circulation caused by liver cirrhosisandtherapidgrowthoftumourcells.Unsurprisingly,liver cirrhosis and tumour size (>8 cm) are both independent predictors of PVTT in HCC. 53 In addition, expression of protein disulfide-isomeraseA6 (PDIA6), apolipoprotein A-I (APO A-I) and CXC chemokinereceptor4(CXCR4)hasbeenidentifiedtocorrelatewith the presence of PVTT, 54,55 and can also be induced by hypoxia. [56][57][58] Importantly, elevated hypoxia-inducible factor alpha (HIF-1α), an important transcription factor involved in the hypoxic response of cells, has been found to closely relate to PVTT and poor prognosis in HCC patients. 59,60 Based on these findings, the association between hypoxia and PVTT formation aroused our interests, and some relatedresearcheshavebeenperformedinourlaboratory.Among143 patients with HCC, we uncovered a causative link between intratumoral hypoxia and PVTT formation. In an analysis of HIF-1α targeted proteins related to HCC progression, we found elevated levels of 14-3-3ζ were induced by hypoxia and correlated with PVTT formation in HCC patients. We then found 14-3-3ζ upregulated HIF-1α expression by recruiting HDAC4, which prevented HIF-1α acetylation, thereby stabilizing the protein. This pro-survival role of 14-3-3ζ in stabilizing HIF-1α was necessary for hypoxia-induced expression of genes, including those indicative of epithelial-mesenchymal transition (EMT), which led to tumour metastasis. 19 Our results estab-  significantly elevated in PVTT tissues and were associated with poor prognosis in patients with PVTT. 23 We previously reported that a

| CIRCUL ATING TUMOUR CELL S AND PV T T
CTCs are cancer cells in circulation dissociated from primary tumours. CTCs can be used as a biomarker to noninvasively monitor cancer progression and guide therapy. 84 There is increasing evidence revealing that CTCs are closely related to recurrence and shorter recurrence-free survival of HCC. 85 PVTT formation. 93 The great efforts are needed to be focused on the molecular mechanisms of CTCs-induced PVTT, which will provide more knowledge about PVTT formation.
GiventhesignificantcorrelationbetweenCTCandEMT,theCTC markers have been assessed in clinical studies as effective biomarkers of HCC. 94 A recent study highlighted that mesenchymal-CTCs gainedmesenchymalfeaturesviaEMTandpromotedHCCmetastasis. 94 They could not only represented the progression and state of HCC, but also serve as a prognostic marker for long-term survivals.

| NON -COD ING RNA S AND PV T T
Non-coding RNAs including miRNAs and lncRNAs are pivotal participants and regulators in the development and progression of HCC. 95 The role of miRNAs and lncRNAs in PVTT formation also has been investigated in the last decade. 17 17 We found an ICR can regulateCSCpropertiesofHCCcellsandICRcontributestoPVTT development. 23 Thus, we believe non-coding RNAs play an im-portantroleinPVTTformationanddevelopment.Uptonow,the regulatory effect of non-coding RNA in the progression of HCC and PVTT has still been under experimental study, further investigationsarerequiredtopavethewayforclinicaltranslation.

| CON CLUS IONS
Clearly, PVTT is common and it worsens prognosis of HCC.
AlthoughamountainofevidenceuncoveredPVTToriginatedfrom the primary HCC tissues, several studies also identified that PVTT may have different clonal origins from their corresponding HCC.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data openly available in a public repository that issues datasets with DOIs.