Epidemiological and observational studies have reported a transition in the trend of the leading major cause of death from cardiovascular diseases to cancer (Hastings et al., 2018; Stringhini and Guessous, 2018). The main reason for such transition is the improvement in early prediction, diagnosis, and treatment of cardiovascular diseases. This raises pressing needs for more research focusing on the early detection of cancer. Invasive female breast cancer (BC) is the most common cancer type and is considered the main cause of death in women every year with approximately 682,000 cases in 2020 (Sung et al., 2021). BC initiation and progression are controlled by a crosstalk of complex regulatory signalling networks which are not yet fully understood. Among these are Wnt, FGF, Notch, Sonic Hedgehog, and BMP signalling (Katoh, 2017).

Wnt ligands and their receptors, Frizzleds (FZDs), are crucial signalling molecules that play a major role in regulating cellular behaviour and gene transcription during embryonic development and in cancer (Wong et al., 2002; Brennan and Brown, 2004; Turashvili et al., 2006; Ueno et al., 2013). This includes proliferation, differentiation, migration, and aggregation (Turashvili et al., 2006). So far, 19 wnt ligands and 10 FZD receptors have been identified in humans. Interestingly, the first two Wnt members (Int-1 and Int-2) were primarily discovered as oncogenes in mouse mammary tumours (Peters et al., 1984; Mester et al., 1987). Expression of at least eight Wnt ligands (Wnt-2, −3, −4, −5A, −7B, −10B, −13, and −14) and several frizzled receptors was reported in different types of liquid and solid tumours including BC (Howe and Brown, 2004; Zhan et al., 2017; Martin-Orozco et al., 2019; Koni et al., 2020; Wu et al., 2020).

During embryonic development, Wnt members expression was shown to play crucial role in the maintenance or specification of the mammary stem cells (Cantilena et al., 2011) and gland ductal formation (Lin et al., 1992; Brennan and Brown, 2004). Interestingly, this expression was found either upregulated or downregulated in cancer (Boras-Granic and Wysolmerski, 2008; Incassati et al., 2010; Yu et al., 2016) indicating that these members could play a dual role in development and cancer. Therefore, they could be potential therapeutic targets in different types of cancer (Yang et al., 2011; Xie et al., 2018; Zeng et al., 2018). Wnt/PCP (planar cell polarity) signalling, which controls the distribution of Wnt/β-catenin in filopodia protrusions (cytonemes), was shown to regulate cancer cell growth by regulating these cytonemes (Mattes et al., 2018; Fereres et al., 2019). This suggests that inhibiting or manipulating Wnt function could lead to identifying potential cancer therapeutic targets. A number of FZD receptors are being tested for antibody therapeutics including FZD1, 2, 5, 7, and 8 in patients with Wnt driven cancers (reviewed in (Katoh, 2017). A better understanding of the functioning of the Wnt/FZDs signalling mechanism is still needed to determine which component(s) should be targeted for efficient biomarker discovery and targeted therapy.

Among the FZD members that have received very little attention in BC is FZD6, despite its important role in other types of cancer including cervical cancer (Wang et al., 2021), colon (Vincan and Barker, 2008; Xu et al., 2019), leukaemia (Wu et al., 2009; Yuan et al., 2019; Cassaro et al., 2021), hepatocarcinoma (Bengochea et al., 2008), squamous cell sarcoma and adenomas (Haider et al., 2006), oral squamous cell carcinoma (Putnová et al., 2021; Sung et al., 2021), neuroblastoma (Cantilena et al., 2011), glioblastoma (Zhang et al., 2021), pancreatic adenocarcinoma (Yang et al., 2019; Li et al., 2021), and prostate cancer (Saramäki et al., 2006; Han K. et al., 2018). Hence, FZD6 was suggested as a promising therapeutic cancer target (Han K. et al., 2018; Zeng et al., 2018; Patel et al., 2019).

Due to the critical role and importance of the Wnt/FZD signalling function as well as the promise of FZD6 as a therapeutic target, we tailored this study to assess FZD6 protein expression in Saudi female BC aiming at unravelling the correlation of its expression pattern with the clinicopathological features and the survival outcome. We, in addition, analysed the possible potential interactions of FZD6 with several microRNAs known to be expressed in BC to further understand their molecular involvement in the biological complexity of the BC.

The word ‘cancer’ is still horrifying to many, but according to WHO it should not be a death sentence to the cancer patient. This will not be accomplished unless we have globally full control of the disease incidence. One way to achieve this is by identifying the cancer inducers using in vivo screening and by discovering new biomarkers that could help us in early diagnosis, prognosis, and therapies. The current study is a part of an OncoScreen project aiming at identifying the cancer inducers as well as early cancer biomarkers.

In Saudi Arabia, BC is the leading cancer type with an incidence of 29.7% in women in 2018. Wnt ligands signal to frizzleds mainly through either canonical (β-Catenin activation-dependent) or non-canonical (Wnt or β-Catenin independent) signalling pathways. The Wnt receptor FZD6 has received, as far as we know, nearly no attention to its role in BC except a very few studies (Vouyovitch et al., 2016; Corda et al., 2017; Poodineh et al., 2020). Remarkably, FZD6 has been reasonably studied in other cancer types such as oral (Putnová et al., 2021), prostate (Vatansever et al., 2014), thyroid (Deng et al., 2015), pancreatic adenocarcinoma (Li et al., 2021), osteosarcoma (De Sá Rodrigues et al., 2017), and leukemia (Wu et al., 2009).

In the current study, we showed that elevated FZD6 expression is strongly associated with the early onset of female Saudi BC patients, tumour invasion, and poor survival outcomes. Most importantly, we showed that a higher FZD6 expression level was significantly associated with the survival outcomes of the BC patients including the recurrence (Disease-Free Survival) and the life expectancy after the primary treatment (Disease-Specific Survival). IPA analysis results showed that FZD6 is implicated in BC, breast adenocarcinoma, and ductal breast carcinoma molecular signalling. MicroRNA TargetScan prediction analysis revealed that FZD6 is a potential target of 30 microRNAs, however, miRabel microRNA prediction platform validated 29 of these microRNA (i.e. except has-miR-302a-b3p). In triple negative BC (TNBC) cell line, miR-130a-3p was shown to block Wnt signalling components among which was FZD6 (Poodineh et al., 2020). In our IPA and miRabel analysis, we did not see this microRNA targeting FZD6, and hence further studies may be required to confirm this finding.

In Saudi female BC patients, it has been shown that expression of the Wnt axis APC/Axin/DKK3/FRP2/WIF1 was downregulated and not associated with the age of onset of the disease (Khan et al., 2018). It has been previously shown that FZD6 promotes TNBC cell motility and metastasis through the fibronectin-actin axis. This suggested that the noncanonical Wnt signalling is involved in basal-like BC/TNBC progression (Corda and Sala, 2017). It has been suggested that FZD6 through this non-canonical Wnt signalling affects the cell motility and cellular invasion (Corda and Sala, 2017), and hence it is considered an important potential therapeutic target (Van Schie and Van Amerongen, 2020). We reported here that FZD6 is associated with the survival outcomes of BC patients. In liver tumourigenesis, FZD6 was the only frizzled gene found to be associated with tumour recurrence and metastasis (Chen et al., 2018). Besides, the association of FZD6 with tumour invasion reported here is consistent with a previous report showing that WNT11/FZD6 were associated with tumour invasion in colorectal cancer (Gorroño-Etxebarria et al., 2019). The association of FZD6 with the tumour metastatic recurrence that we showed in our current study is also consistent with the previous study mentioned above in TNBC (Corda, 2015). In cervical cancer, silencing FZD6 function caused delayed cellular proliferation, invasion, and EMT transition through HOXC13/WNT5A/FZD6 axis (Tongfei et al., 2021). Similarly in our cohort, cell proliferation, invasion and EMT could be driven by FZD6 elevated expression.

In our IHC analysis, FZD6 did not show a significant correlation with known BC prognostic markers ER/PR or HER2 (Table 1). Our knowledge-based IPA analysis did not also show if these markers were ‘direct’ upstream or master regulators of FZD6. This suggests that FZD6 probably functions ‘indirectly’ of these markers. In addition, there were some studies that reported some examples of elevated expression of BC biomarkers with either no correlation with ER expression, such as Endoglin (Guo et al., 2017), or not statistically significant in ER-positive BC such as TOX3 protein expression (Han et al., 2016). For the HER2, the worldwide prevalence of its amplification in BCs ranges only between 15 and 30% while in other types of cancer, such as colorectal cancer, HER2 does not show a prognostic value (Kruszewski et al., 2010). The heterogeneity and multiclonality of BC influenced by the population genomic background, patients’ lifestyle and the environmental risk factors is making BC a challenging and biologically complex disease with unexpected correlations and outcomes.

Fluorescence recovery after photobleaching (FRAP) approach showed that several Wnt ligands including WNT-1, -2, -3A, -4, -5A, -7A, -9B, and -10B bind to Fzd6 (Kilander et al., 2014). It is always the question of which WNT ligand could initiate FZD6 in BC. The validated function of the Wnt ligands (WNT 2, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 9a, 10a, 10b, and 11) and their receptors (FZD1, 2, 6, and 7) in BC were recently reviewed in some detail by Xu and his colleagues (Xu et al., 2020). In breast cancer cells and in solid autocrine human growth hormone (hGH) tumours, both WNT4 and its receptor FZD6 were upregulated (Vouyovitch et al., 2016) indicating that WNT4 is a strong Wnt ligand candidate to activate FZD6. Our in silico analysis using IPA showed that Wnt5a and Wnt16 are the most aberrant WNT ligands to signal to FZD6 in BC (Table 3), however, these results need further validation. It is worth mentioning that Wnt5a/5b are involved in BC invasiveness and metastasis independent of β-catenin signalling (Klemm et al., 2011; Han B. et al., 2018).

The IPA analysis revealed a considerable number of putative microRNAs that could potentially act to silence FZD6 function. We validated these microRNAs using TargetScan and pulled 30 members as potential miRNAs that could target FZD6 function in BC (Table 5). We further used miRabel database and validated all these microRNAs except one microRNA. A considerable number of these microRNAs have been shown to have a pivotal role in BC regulation (Tsai et al., 2018). Among these, as an example, is mir-302b, a microRNA we have identified in our IPA analysis, which was shown to target FZD6 in oral squamous cell carcinoma to promote cell invasion and migration (Sun et al., 2021). It was also previously shown that miR-199b-5p targets HER2 in BC (Fang et al., 2013) as well as directly targeting Fzd6 to activate the signalling cascade of Wnt4, β-catenin, Tcf7, and C-myc during thymic aging (Wang et al., 2020).

The above-mentioned findings we reported here suggest a poor prognostic value of FZD6 overexpression in the early onset of BC through probably affecting cell proliferation, EMT, distant metastasis, and by compromising the normal molecular signalling cascade involved in these processes. Further experimental validation of FZD6 master regulators including microRNAs is needed with taking into consideration other published data that are not included in our analysis.

The current study is a part of a cancer prevention program called OncoScreen aiming at screening for cancer inducers and identifying biomarkers for early cancer diagnosis and prognosis. Expression pattern analysis of FZD6 in a Saudi BC cohort showed that its elevated expression is associated with tumour invasion, metastasis, and worse survival outcomes mainly in younger patients. Several WNT ligands and microRNAs were shown to potentially regulate FZD6 expression and function. As far as we know, this study is the first to analyse FZD6 expression in female BC Saudi patients and assess its prognostic value.