Dysregulated expression of slingshot protein phosphatase 1 (SSH1) disrupts circadian rhythm and WNT signaling associated to hepatocellular carcinoma pathogenesis

Introduction

Liver cancer is the sixth most diagnosed malignancy and third most common cause of cancer-related mortality worldwide, with 905 677 new cases and 830 180 deaths in 2020 alone [1, 2]. Of the histological types of liver cancer, namely, cholangiocarcinoma (CC), focal nodular hyperplasia (FNH), hepatocellular adenoma (HCA), hepatocellular carcinoma (HCC), and combined hepatocellular carcinoma - cholangiocarcinoma (HCC-CC) [3], HCC accounts for about 75%, is mostly associated with chronic hepatic disease including liver cirrhosis, hepatitis B and hepatitis C [1, 2, 4]. Coupled with the rise in the annual number of new cases, patients with HCC are characterized by poor survival rates, such that the median survival time of inoperable cases ranges between 6 and 20 months, with a 5-year survival rate less than 5% [1, 2]. This may not be unconnected with patients presenting with advanced disease, vascular invasion, multifocal or multicentric large tumor, concomitant morbidities, or even organ failure [5].

Metastasis, a hallmark of cancer, remains a principal cause of cancer-related deaths, accounting for about 90% of all such deaths [6]. Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSCs) are two major interrelated contributors to the metastasis of malignant cells [6, 7]. CSCs, originating from de-differentiated cells or tissue-resident stem cells, are a subset of malignant cells in the tumor bulk that inherently drive tumorigenesis, self-renewal of cancer cells, and cancer recurrence after initial treatment [6–8]. The accruing evidence indicating that cancer stemness is a reversibly acquired or lost cell state, and the phenotypic plasticity of CSCs, further accentuates the inter-relatedness of CSCs phenotypes and EMT, where the latter reflects the ability of cancer cells to switch cellular states [8, 9]. The activation of EMT is increasingly associated with the acquisition of CSCs-like traits, implicated in various cancer types, and induces resistance to contemporary chemotherapeutic agents [9–11]. EMT entails the loss of cellular apicobasal polarity, cytoskeletal rearrangement, and the acquisition of mesenchymal gene signature [9–12]. This transient loss of junctional complex proteins and acquisition of mesenchymal proteins facilitates the development of phenotypically more aggressive quasi-mesenchymal cell states in malignancies, including HCC [11–13]. The EMT-associated induction of actin structures and reorganization of cellular actin cytoskeleton facilitates enhanced cell migration and invasion [10–12]. The migration of malignant and non-tumor cells is a vital component of metastasis and depends on changes in the actin cytoskeleton typified by lamellipodia, filopodia, and invadopodia formation. These changes in the actin cytoskeleton are regulated by several actin-binding proteins such as cofilin-1 and slingshot protein phosphatase 1 (SSH1; also known as cofilin phosphatase or slingshot homolog 1) [14, 15].

The intricate interplay of biological processes in the human body is a testament to its complex regulatory mechanisms. At the heart of these mechanisms is the circadian rhythm, a natural, internal process that regulates the sleep-wake cycle and repeats on each rotation of the Earth roughly every 24 hours [16]. This rhythm does more than dictate our sleep patterns; it has an influential role in a multitude of physiological processes including hormonal secretion, cellular function, and metabolic regulation [16].

The intriguing intersection between SSHs and the circadian rhythm is an emerging area of research. Protein phosphatase 1 (PP1) is an example of a post-translational regulator of the mammalian circadian clock [17]. The slingshot family protein phosphatases (SSHs) currently compose of structurally overlapping but functionally distinct SSH1, SSH2 and SSH3 in mammals, and are reported to ‘specifically dephosphorylate and reactivate Ser-3-phosphorylated cofilin (P-cofilin)’, where upregulation of cofilin-1 has been shown to positively correlate with cancer aggression and metastasis [15]. SSH1 is activated by F-actin and the cofilin–phosphatase activity of SSH1 is strongly increased by F-actin binding, however, SSH1 exhibits two seemingly diametric activities in actin filament dynamics, namely, a cofilin–phosphatase activity that induces F-actin disassembly through cofilin-1 activation, and cofilin–phosphatase activity-independent stabilization and bundling of F-actin [18]. Despite this background understanding, if, how and to what extent SSH1 is involved tumor initiation, cancer metastasis, resistance to therapy, and poor prognosis remain largely under-explored, and more so compounded by our limited understanding of how the seemingly opposing actions of SSH1 are controlled in the context of the HCC microenvironment.

Hepatic homeostasis, a balance in the liver’s physiological processes, is essential for maintaining health. The liver, being a crucial metabolic organ, has many time-sensitive functions and, as such, is governed by the circadian rhythm [19]. This includes processes such as glucose metabolism, lipid processing, and detoxification pathways. Disruptions in this timing can lead to hepatic disorders and are often seen in conditions like fatty liver disease, cirrhosis, and liver enzyme abnormalities [19, 20]. Hepatocellular carcinoma (HCC), the primary form of liver cancer, is a culmination of numerous hepatic dysfunctions. The emerging body of research now suggests that circadian rhythm disturbances can influence the pathogenesis and progression of HCC [21]. There’s a growing understanding that aberrations in the circadian genes and proteins could play a role in liver tumorigenesis [22]. Additionally, factors that disrupt hepatic homeostasis, like metabolic disorders and inflammation, might act synergistically with circadian disruptions to enhance HCC risk. The existence of a temporal coordination of actin regulators has recently been suggested to drive cell-intrinsic actin dynamics, such that a presumed cellular clock controls and/or determines the efficiency of actin-dependent processes including cell adhesion and migration, thus ultimately impacting the efficacy of wound healing [23]. Since the ability of malignant cells to metastasize to other sites or organs depends on their ability to pervert and exploit natural wound-healing pathways [24], hypothesizing that SSH1 plays a vital regulatory role in the circadian modulation of the cellular actin cytoskeleton and that this influences cell motility, propensity to metastasize, treatment response, and ultimately the prognosis in patients with HCC.

The present demonstrated that SSH1 is significantly upregulated in HCC clinical samples, compared to their non-tumor counterpart and this aberrant expression of SSH1 is associated with worse overall and progression-free survival rates. We showed that pharmacological or CRISPR inhibition of SSH1 in HCC cell lines significantly suppressed cell viability, migration, invasion, colony and tumorsphere formation capabilities of the HCC cells. Mechanistically and of significant interest, we found that the molecular or pharmacological inhibition of SSH1 elicited suppression of CLOCK (circadian locomotor output cycles protein kaput), ARNTL/BMAL1 (aryl hydrocarbon receptor nuclear translocator like), WNT3 (Wnt family member 3), β-catenin, LRP5/6 (low-density lipoprotein receptor-related protein 5/6), BCL2 (B-cell CLL/lymphoma 2), vimentin (VIM) and Snail, with concomitant upregulated expression of cofilin-1/2 (CFL-1/2), and Cryptochrome (CRY), indicating dysregulated circadian rhythm and deactivation of the WNT/β-catenin oncogenic pathway. Consistent with these findings, we posit that the interplay between SSH1 and the ‘clock genes’ plays a critical role in actin cytoskeleton dynamics, the acquisition of cancer stemness phenotype, augmentation of cancer aggressiveness, HCC progression, and poor prognosis.

Results

Discussion

The formation and progression of HCC is characterized by changes in the gene expression profile of hepatocytes, and this consequently leads to the acquisition of malignant, aggressive, and often therapy-resistant phenotypes observed in cancerous hepatic cells [3–6, 10, 25]. Several studies have suggested that SSH1 may play some critical roles in the maintenance of protein structure and stability, regulation of cellular localization, function and activities, as well as the homo- and hetero-dimeric protein-to-protein interactions, with an apparent dearth of information on its complicity in HCC [14]. Over the last decade, there has been reports suggesting that cell migration, an essential component of metastasis typified by altered actin cytoskeleton such as lamellipodia, filopodia, and invadopodia formation (regardless of their cancerous status), is regulated by certain actin-binding proteins including the cofilin phosphatase SSH1 [15].

Recently, there have been reports indicating that upregulated SSH1 expression was significantly associated with metastasis and strongly correlated with poor prognosis in patients with gastric and colorectal cancers [26]. Consistent with these reports, the present study demonstrated, for the first time to the best of our knowledge, that SSH1 is aberrantly expressed in HCC and that this overexpression of SSH1 is prognostic of poor clinical outcome in patients with HCC (Figures 1, 2). The clinical significance and statistical relevance are evident when the Kaplan-Meier plots indicate that patients exhibiting high SSH1 levels face a heightened risk of mortality and disease progression. The utility of receiver operating characteristics (ROC) curves in disease diagnosis and prognosis cannot be overstated. Their application is especially pertinent when discerning the discriminative ability of biomarkers like SSH1 or when validating its role as a diagnostic and prognostic indicator. In the realm of biomarker identification and validation, the Area Under the Curve (AUC) is a renowned measure of the overall accuracy of discrimination. A higher AUC value signifies the superior discriminatory prowess of a given diagnostic or prognostic tool [27]. Within this study, the observed AUC ranged from 0.62 to 0.77, which is satisfactory, underscoring the importance of SSH1 as a pivotal biomarker. This observation is further corroborated by subsequent experiments.

Our data showing that the molecular inhibition or pharmacological targeting of SSH1 suppressed the viability, oncogenicity, and cancer stemness of HCC cells (Figure 3), is also of clinical relevance, and in line with similar findings in colorectal cancer, wherein the shRNA-mediated loss of SSH1 significantly impaired the migration, invasion, and colony formation of colorectal cells, in vitro [28]. Cancer stem cells (CSCs) are intrinsically self-renewing, and as such their presence or enrichment confer the so-called cancer stemness phenotypes, which are increasingly implicated in resistance to metastasis, chemotherapy, and cancer recurrence, which continue to constitute a major clinical challenge to effective anticancer treatment, especially as an effective CSC eradication strategy remains elusive [6–11, 29]. It is thus noteworthy that targeting SSH1 strongly suppressed cancer stemness phenotypes, including colony and tumorsphere formation by HCC cells, and may be exploited for the development of a CSCs elimination-based anticancer therapeutic strategy for patients with HCC.

It is also of therapeutic relevance that we found that the oncogenic activity of SSH1 is mediated by circadian rhythm disruption and Wnt/β-catenin signaling activation (Figure 4). Consistent with these findings, significantly reduced tumor volume was observed in the mice treated with Sennoside A (p < 0.01). The expression of SSH1, CLOCK, BMAL1 and β-catenin proteins were significantly downregulated in the Sennoside A_dark treat mice (Figure 5), indicating that the oncogenic activity of SSH1 has a circadian rhythm molecular underlining and that this can be exploited for the therapeutic benefit of patients with HCC. There is evidence that aberrant Wnt/β-catenin signaling facilitates the renewal of CSCs, proliferation and differentiation of cancer cells, thus playing a vital role in tumor formation, cancer progression, and therapy resistance [30]. This vital role informs continued research efforts to demystify this Wnt/β-catenin signaling, identify its activators, and in so doing, develop specific and targeted antagonists or inhibitors of this oncogenic pathway as effective therapeutic strategies for personalized anticancer treatment. Our findings indicating that the oncogenic activity of SSH1 is mediated by circadian rhythm disruption and Wnt/β-catenin signaling activation is consistent with recent reports that low levels of the period circadian regulator 3 (PER3) induces the BMAL1 expression, leads to β-catenin phosphorylation and activation of the WNT/β-catenin signaling pathway, and is characteristic of ALDH^hiCD44⁺ prostate CSCs [31]. This further emphasizes the clinical benefits of targeting SSH1 in malignant diseases, including HCC.

To reiterate, the circadian rhythm, often referred to as the circadian clock, adjusts, shapes, and synchronizes external stimuli and environmental shifts with the organism’s internal processes [23, 32]. In essence, this clock ensures the organism’s timely accuracy and strong adaptability to its environment. Disruptions or misalignments in the circadian rhythm can elevate the risk of systemic illnesses, including cancer [32]. Our current research indicates that when SSH1 expression is decreased, there is a suppression in the ‛clock proteins’ CLOCK, BMAL1, and metastasis markers like WNT3, β-catenin, LRP5/6, BCL2, VIM, and Snail. Concurrently, there’s an increase in ‛clock proteins’ CFL-1/2, and CRY1. These findings enrich our understanding of how an imbalanced circadian rhythm can fuel cancer-causing signals. They also underscore the potential of targeting circadian signals as a promising clock-based treatment approach for HCC patients. These insights bolster the emerging field of circadian medicine. Within this domain, therapeutic methods, such as SSH-/- or Sennoside A, which focus on central clock components and their immediate regulators to modify the circadian rhythm, are being refined to maximize their therapeutic efficacy [32]. Moreover, the impact of SSH1 can be incorporated into HCC treatment strategies through circadian rhythm modulation. Nonetheless, additional research is required to reaffirm and establish a significant correlation between SSH1 and downstream signaling in HCC.

In conclusion, as depicted in Figure 6, our study highlights that SSH1 serves as an actionable molecular biomarker associated with actin-binding, stemness, and circadian rhythm modulation. Its therapeutic and prognostic potential for HCC patients is notably significant.

Materials and Methods

Supplementary Materials

Supplementary Figures

Supplementary Table 1