20p12.3-p12.2

ARCND2,PI-PLC

Since the beginning, given the link of the Drosophila homologous gene norpA with altered photo-transduction and retinal degeneration (Bloomquist BT et al. 1988), PLCB4 was suspected of being responsible for a similar pathology in humans. So far it has not been shown that alterations of PLBC4 are responsible for a precise nosological entity affecting human retina, though there is proof of the importance of the gene in the physiology of vision (Jiang H et al. 1996). Unexpectedly, the gene was found mutated in the Auriculo-condylar Syndrome (Rieder MJ et al, 2012), a cranio-facial malformation characterized by mandibular-maxillary homeotic transformation. These extremely rare, de novo PLCB4 mutations lead to an impairment of the endothelin-1-distal-less homeobox 5 and 6 ( EDN1 -DLX5/DLX6) pathway, known for its importance in mandibular development of other species. PLCB4 is currently known as an important gene for the development of the first and second pharyngeal arches in embryo.
Phosphoinositides are chemically phospholipids and are the specific substrate of all PLCs, including PLCB4. Phosphoinositides have an established role in cancer development as they can affect proliferation, survival and spreading of cancer cells. The two second messengers produced by PLCB4, InsP3 and DAG, are responsible for the Ca⁺⁺ increase in the cytosol and the activation of Protein Kinase C (PKC) respectively. Both are factors capable of influencing neoplastic growth. PIs (phosphoinositides) and their metabolic enzymes modulate apoptosis, proliferation, differentiation, vesicular trafficking, cell adhesion, and cell migration (Owusu Obeng et al., 2020).

Uveal melanoma

Uveal melanoma (UM) is the first intraocular malignancy by frequency and is affected by poor prognosis with a 50% metastatic rate (Afshar AR et al, 2019). In 2015 (Johansson P et al., 2015) PLCB4 was added to the list of driver genes for UM ( GNAQ, GNA11, CYSLTR2, EIF1AX, SF3B1, BAP1). PLCB4, GNAQ and GNA11 are G-protein pathway-associated and they belong to the same Gαq pathway. A recurrent mutation (Asp630Tyr) in the sequence of Y domain was found in 2 out of 28 samples by whole genome sequencing (n=14) or whole exome sequencing (n=9). Cases with a range of different mutations affecting the same codon D630 (Asp630) have been reported in the medical literature (Asp630Val; Asp630Fen; Asp630Asn) (Johansson P et al., 2015). PLCB4 is normally activated by GNAQ interaction (PLCβ4 is the downstream effector of Gαq signaling) (Moore AR et al., 2016). Considering that the mutations in UM are mutually exclusive, it can be inferred that PLCB4 hotspot mutation is a recurrent gain-of-function mutation activating the same pathway involving GNAQ/GNA11. Mutations of the genes of this pathway account for 98% of UM cases. PLCB4 mutation frequency is 2,5% and it could be a target for specific inhibitors (Chua V et al., 2017). In order to counter the PLCB4 activity the PKC-inhibitor LXS196 has been tested with partial responses in metastatic UM (Violanti SS et al., 2019). It is remarkable that derangement of the Gαq signaling (heterotrimeric G proteins) is specific for UM and not for cutaneous malignant melanoma (CMM). PLCB4 D630Y (Asp630Tyr) mutation is rarely detected in primary leptomeningeal melanocytic tumors (PLMTs or "melanocytomas"). This mutation helps in the differential diagnosis between PLMTs and metastasis to central nervous system by cutaneous malignant melanoma. PLCB4 D630Y (Asp630Tyr) is always absent in cutaneous melanoma and its metastases and this differentiates the two tumors, which is clinically relevant given the frequently benign nature of the PLMTs (van de Nes et al., 2017).
A very recent observation has drawn attention to the causal link between ocular melanocytosis and UM. Ocular melanocytosis strongly predisposes to UM (Singh AD et al. 1998) with risk of UM increased from 1 in 230,000 to 1 in 400. A case report focusing on this clinical correlation (Durante MA et al., 2019) refers to a patient affected by UM arose from a pre-existing ocular melanocytosis. Slightly less than 25% of the melanocytosis cells and 100% of the melanoma cells revealed to be affected by PLBC4 D630Y in the absence of germinal mutation. Genetic complete characterization showed that PLCB4 mutation was the initiating, but not the transformation factor in the described case report. In the context of melanocytosis a condition of loss of heterozygosity arising in the chromosome 3 likely represents the "threshold event" on the way to malignant transformation of the PLCB4-mutated clone.

Acute Myeloid Leukemia (AML)

AML, together with brain tumors, holds the primacy among the causes of infant death worldwide. Survival has improved in recent years (Curtin SC et al, 2016) but the overall survival (OS) is still relatively low and there is a need to identify molecular parameters of prognostic and therapeutic significance. This is the purpose of a recent study (Wu S et al., 2019) that investigated the prognostic value of PLCB4 overexpression in pediatric leukemia.

In silico analysis of expression profiles obtained from the database TARGET on a sample of 285 pediatric de novo AML (pAML), classified patients as low-PLCB4 and high PLCB4 group. Overall it emerged that PLCB4 overexpression independently predicts a poor prognosis in case of chemotherapy but not in case of stem cell transplantation (SCT) in CR1 (complete remission after first cycle).
Survivors at 5-years follow-up had a median value of PLCB4 mRNA expression significantly lower with an overall survival of 60.7 months vs 28.5, and an event-free survival of 16.3 months vs 12.5. Patients with long-term CR and lower relapse rate had a median value of PLCB4 mRNA expression significantly low. Moreover, white blood count correlates positively with PLCB4 expression observing the highest PLCB4 expression in CD34+CD38- cells compared to CD34-CD38+ or CD34+CD38+ cells (Wu S et al., 2019). These results are confirmed by other study (Zheng GH et al, 2009) who found upregulation of PLCB4 in an AML cell-line (HL-60/MDR) with multi-drug resistance. Furthermore, there are other studies where PLCB4 appears to behave in the opposite way, for example in the case of PLCB4 upregulation manifested by breast cancer patients with complete response to chemotherapy (Li Y et al., 2017).

T-Acute Lymphoblastic Leukemia (T-ALL)

In addition to AML there are hints that PLCB4 may be involved as well in pediatric T-ALL (Haider Z et al., 2019). A set of 216 genes was overexpressed in CIMP-negative (CpG Island methylator phenotype) subgroup, characterized by a worse prognosis in T-ALL (Borssen M et al., 2013), while 548 genes were overexpressed in CIMP+. PLCB4 shows significantly higher expression in CIMP+ T-ALL, associated with better prognosis, and this is true even when compared to normal T cells. No in-depth analysis was carried out in this study regarding the molecular mechanism of this overexpression and its possible consequences on the cell fate.

Breast Cancer

Primary operable breast cancer undergo neoadjuvant chemotherapy (NST = Neoadjuvant Systemic Therapy) as a standard option. The response to NST can give information on the likelihood of cancer recurrence and survival, in this way the best chance would be to have predictors of NST response because patients with low probability of NST response could be spared unuseful toxic treatment. Various biological markers have been investigated for this purpose such as negative hormone receptors status, ERBB2 (Her2/Neu) positivity, TOP2A (Topoisomerase IIα), BCL2 loss (Kaufmann M et al., 2007).
Studies were conducted to define a gene expression profile linkable to NST response. Li and coworkers analyzed two small groups (3 vs 3) of breast cancer patients on the base of complete or not-complete pathologic response (NST-responding or NST-non responding) to chemotherapy (Li Y et al., 2017). A set of 673 differentially expressed genes (DEG) emerged and a pathway enrichment analysis was performed. Results identified a set of 18 genes, including PLCB4, that effectively stratified the sample in responders and non-responders. Four out of these 18 genes are considered key genes on the base of their protein-protein interaction pattern (PLCB4, ADCY6, CNR1, MAPK14). In particular upregulated PLCB4 is positively involved in pathological complete response to chemotherapy, intervening in multiple pathways (renin secretion, gastric acid secretion, gap junction, inflammatory mediator regulation of TRP channels, retrograde endocannabinoid signaling, melanogenesis, cGMP-PKG signaling pathway, calcium signaling pathway, chemokine signaling pathway, c-AMP signaling pathway, rap1 signaling pathway). At least two other isozymes of the PLCB family ( PLCB1 and PLCB2) are reported as possible prognostic markers (Cai S et al., 2017). Overexpressed PLCB4 and PLCB2 have been associated with multidrug resistance (MDR) in breast cancer. The identification of differentially expressed genes between the breast cancer cell lines MCF-7 and MCF-7/MDR cells confirmed PLCB4 as an overexpressed hub gene (Yang M et al., 2018).
PLCB4 is one of the primary target genes of TFAP2C, an important transcription factor playing a role in the oncogenesis of breast cancer. In mammary tumors TFAP2C induces a very large number of genes belonging to different functional groups, including a set of intracellular signaling genes such as PLCB4 (Woodfield GW et al. , 2010).

Colon adenocarcinoma (COAD)

There are molecular differences between right colon adenocarcinoma (RCOAD) and left colon adenocarcinoma (LCOAD), RCOAD has a worse prognosis than LCOAD (Han J et al., 2020).
Genomic data of 9 cases of RCOAD and 9 of LCOAD have been recovered from open-source platform GEO (Gene Expression Omnibus-GEO database), selection of DEGs (differentially expressed genes) between these two groups of colon cancers identified 286 DEG genes. Construction of protein-protein interaction network of the DEGs and identification of hub genes have been performed. PLCB4 resulted downregulated in RCOAD compared to LCOAD. A gene set was therefore able to differentiate between RCOAD and LCOAD. Despite the fact that PLCB4 belongs to the hub gene group and is markedly downregulated in RCOAD no association with pathological stage nor statistically significant effect on overall survival have been demonstrated and this excludes a causal role of PLCB4 in the worse prognosis of RCOAD vs LCOAD.

Non-small cell lung carcinoma (NSCLC)

Overexpression of PLCB4 has recently been associated with a better prognosis of lung adenocarcinoma. A in silico study (Zhang T et al., 2019) analysed a cohort of 1926 NSCLC (non-small cell lung carcinoma) patients starting from gene expression and survival values. It emerged that overexpression of PLCB1, PLCB2, and PLCB3 are related to poor overall survival of all NSCLC patients and poor prognosis of adenocarcinoma. On the contrary PLCB4 overexpression improves the OS of adenocarcinoma patients. Of interest, the correlation analysis of PLCB family found that these four genes are linked to each other in a complex network. Conclusions suggested that not only PLCB4 but the PLCB subfamily as a whole has a possible use in defining the prognosis of the NSCLC patients.
Disruption of Gq signaling (the pathway including PLCB4) by a competitive inhibition mechanism reduces the basal activity of PKC and promotes inhibition of small cell lung cancer growth (Beekman A et al, 1998).

Mesothelioma

Evidences on the relationship between mesothelioma and PLCB4 derives from the study of the effect of PLCB4 knockdown in mesothelial cell lines (Kakiuchi T et al., 2016).
Comparative gene expression analysis reveals overexpression of PLCB4 in YAP^S127A-transformed cells vs control cells (Kakiuchi T et al., 2016). Moreover YAP knockdown (Mizuno T et al., 2012) downregulates PLCB4 in Hippo-disrupted mesothelioma cell lines and PLCB4 knockdown inhibits the cell growth in 8 mesothelioma cell lines (4 YAP-active and 4 YAP-non active) only if they are YAP active. These effects are evidence of the activating function of YAP on its downstream effector gene PLCB4. PLCB4 is critical for proliferation and anchorage- independent growth of YAP-dependent mesothelioma cells and represents a potential pharmacological target in YAP-active mesotheliomas. In addition to Hypo other pathways are involved in mesothelioma molecular mechanisms such as Wnt (Guo G et al., 2015) and Ras/mitogen activated protein kinase. Wnt can be affected by NF2 (neurofibromin 2) knockdown and dasatinib, a tyrosine kinase inhibitor antagonizing the YAP- CTNNB1 (β catenin)- TBX5 complex, impairs growth of NF2-knocked cells (Kakiuchi T et al., 2016).

Glioblastoma

In consideration of the finding that copy number loss commonly affects genes involved in phosphoinositide signaling pathway, e.g. PTEN and PK3C2B (Kim YW et al., 2013; Gu Y et al., 2013) a number of different phosphoinositide pathway genes (45 genes) were investigated by in silico analysis. Copy number amplification of the chromosome 20, that carries PLCB4 and its isoform gene PLCB1, was detected in 30% of the total 638 tumour samples analysed. STRING software approach confirms that phosphoinositide pathway gene products shows high interaction level. Loss or gain of one gene can change the pattern of the phosphoinositide network as a whole. The conclusion is suggested that general analysis of the phosphoinositide pathway can shed light on tumoral biochemical pathways to drug resistance (Waugh MG, 2016).

Gastrointestinal stromal tumor (GIST)

The search for potential new molecular targets is justified by the very frequent phenomena of resistance that occur in the therapy of GISTs through Imatinib administration (Debiec-Rycther M et al., 2005). A set of 77 genes involved in lipid catabolic processes was subjected to in silico screening by analysis of a population of 350 GISTs. PLCB4 resulted the top-ranking gene being overexpressed in 30% of total cases. Association of overexpression with high-risk cases and presence of metastasis was very strict. Moreover, PLCB4 copy number variations (amplification or polysomy) results in PLCB4 overexpression in 50% of cases (Li CF et al., 2017). PLCB4 copy gain and protein overexpression are independently a robust marker of shorter DFS (disease free survival) and worse prognosis in which amplification has a more negative impact than polysomy. The fact that around half of the cases with PLCB4 overexpression lack copy number gain still requires an explanation. In this viewpoint a molecular mechanism alternative to copy number amplification could be the positive regulatory effect of YAP1 on PLCB4. Accordingly, YAP1 knockdown significantly reduces PLCB4 mRNA and PLCB4 protein expression. YAP1 knockdown and silencing of PLCB4 by RNA interference both induce proliferative slowdown of GIST cells. Copy gain and protein overexpression being not equivalent prognostic markers it is advisable to complement PLCB4 immunostaining with PLCB4-specific FISH assay.

Hepatocellular carcinoma (HCC)

HCC is a poor prognosis cancer with a 12% rate of 5-years survival. There is need of innovative biomarkers with relevance in prognosis, early diagnosis and targeted therapy. The marked structural and functional similarities of isoenzymes belonging to the PLCBs subfamily justify their collective analysis in a recent HCC study (Wang X et al., 2019). Molecular genetics and clinical data was obtained from a public dataset including 212 patients. PLCB4 was expressed at the same level in tumor and normal tissue although transcriptional levels were higher in cancer cells. PLCB4 was found to have no diagnostic significance unlike the other members of the subfamily (PLCB1 and PLCB2 more than PLCB3). Only PLCB1 expression was negatively associated with OS and RFS. The other 3 genes of the subfamily, PLCB4 included, did not affect the prognosis.
This study seems to suggest that more interesting results can be obtained by studying the PLCB gene network rather than individual members alone.

Hemangioblastoma (HB)

HB is an uncommon vascular tumour of benign nature affecting the central nervous system. The familial subtype of HB occurs in Von Hippel-Lindau disease and the typical mark of this tumour subtype is a germinal alteration of the VHL gene. Different genetic anomalies contribute to the HB genesis due to the fact that only 4-25% of sporadic case are associated to a VHL gene somatic mutation. A whole-exome study explored the existence of additional genetic alterations in a sample of 11 patients, six affected by a sporadic form and five affected by a familial form (Ma D et al., 2017).
270 somatic single nucleotide variations (SNV) and a large number of copy number variation (CNV) were found. The observation that many of the genes harboring CNVs are members of different tumour-related pathways suggests that they can somehow influence the disease susceptibility. In sporadic tumours five genes, including PLCB4, were affected by CNVs. In familial tumours three genes had CNVs. In particular PLCB4-copy number deletion was present in 2 patients and amplification in 4 patients. Possible underlying mechanisms relating PLCB4-CNVs to HB tumorigenesis was not investigated.