PRR11 in Malignancies: Biological Activities and Targeted Therapies

Proline rich 11 (PRR11), initially renowned for its relevance with cell-cycle progression, is a proline-rich protein coding gene in chromosome 17q22-23. Currently, accumulating studies have demonstrated that PRR11 plays a critical role in cellular proliferation, colony formation, migration, invasion, cell-cycle progression, apoptosis, autophagy and chemotherapy resistance via multiple signaling pathways and biological molecules in several solid tumors. In particular, PRR11 also serves as a promising prognostic indicator in a limited number of human cancers, gradually manifesting its potential application for targeted therapies. In this review, we summarize functional activities, related signaling pathways and biological molecules of PRR11 in various malignancies and generalize potential application of PRR11 for targeted therapies, thereby contributing to further exploration of PRR11 in cancer treatment.


Introduction
Proline rich 11 (PRR11), situated in chromosome 17q22-23, is a proline-rich protein coding gene with ten exons and nine introns [1,2]. It is adjacent to spindle and kinetochore associated 2 (SKA2), sharing a NF-Y-regulated bidirectional promoter, which could be precisely modulated by p53 [3]. The PRR11 mRNA is 2408 bp in length, containing an open reading frame encoding 360 amino acids [2]. At the protein level, PRR11 is composed of a nuclear localization signal (NLS), two proline rich regions (PRs) and a zinc finger domain (ZFD) [4] (Figure 1a,b).

Tongue Squamous Cell Carcinoma (TSCC)
TSCC, identified as the most common type of tongue cancers, has a propensity of regional recurrence and lymphoid metastasis [23,24]. Though the standardized chemotherapy of cisplatin for TSCC patients would bring survival amelioration, the emergence of chemo-resistance is inevitable, ultimately leading to functional defects [24,25]. Thus, reliable therapeutic targets are essential for improvement of clinical efficacy in TSCC patients. According to assessment by qRT-PCR and Western blot, PRR11 expression is dramatically augmented in TSCC specimens and several TSCC cell lines, including Cal27, SCC15, HSC3, HSC4, HSC6, UM1 and UM2, relative to the normal oral keratinocytes (NOKs) and equivalent non-cancerous tissues [8,22]. Given the clinical characteristics and immunohistochemistry of these TSCC patients, PRR11 serves as an independent prognostic factor for TSCC patients via correlation with the clinical stage, TNM classification, vital status and overall survival [22]. Subsequent in vitro experiments illustrated that PRR11 overexpression significantly enhanced cellular proliferation, invasion and cell-cycle progression through downregulation of p21 and p27 and upregulation of cyclin-dependent kinase 2 (CDK2) and cyclin A2 in TSCC cells. In addition, an in vivo subcutaneous tumorigenicity assay also found that PRR11 knockdown diminished the tumor size and Ki-67 expression in TSCC tissues [8]. Above all, PRR11 serves as an oncogene in TSCC patients, which could be utilized for targeted therapies.

Esophageal Squamous Cell Carcinoma (ESCC)
ESCC, accounting for 90% of esophageal carcinoma, has the highest occurrence in East Asian countries [26,27]. Due to its metastasis, recurrence and lack of comprehensive treatment, the overall five-year survival rate of ESCC patients is still dismal, with a proportion of roughly 15% [26,28]. Therefore, exploring novel diagnostic indicators would be of clinical benefit to ESCC patients. Analysis of ESCC patients from the GEO database has verified the oncogenic role of PRR11 in ESCC tumorigenesis [29]. Relatively higher expression levels of PRR11 are also detected in 12 ESCC cell lines and 38 ESCC tissues than in 2 normal esophageal epithelial cells (NEECs) and the matched adjacent non-tumor tissues [4,15]. Further investigation revealed that PRR11 dramatically facilitated cellular proliferation and the migratory and invasive capacities of ESCC cells targeting Akt and EMT signaling via collaboration with SKA2 [4]. Aiming at cancer stem cells (CSCs), PRR11 knockdown reduced CSC-like phenotypes and tumorigenicity of ESCC cells in vitro and in vivo through Wnt/β-catenin signaling [15]. Surprisingly, chalcone, with chemical modification by dithiocarbamate scaffolds, suppressed the cell growth, migration and invasion of ESCC cells targeting PRR11 [30], showing great potential application for targeted therapy of ESCC patients. However, there is no evidence in evaluating the connection between PRR11 expression and clinical features of ESCC patients. In summary, PRR11 might represent a novel prognostic marker for ESCC patients, which needs further clinical verification.

Non-small Cell Lung Cancer (NSCLC)
NSCLC, the most common histopathological classification of lung cancer, has become the main cause of cancer-associated deaths in urban populations [31]. Though compre-hensive treatment has developed in recent years, earlier metastasis usually occurs when initially diagnosed, leading to poor prognosis [32,33]. Thereby, there is an urgent demand to develop novel targets for NSCLC patients. Compared to 8 normal tissues, the expression level of PRR11 in 40 NSCLC tissues is obviously upregulated. Further investigation concerning clinical features of NSCLC patients revealed that PRR11 contributes to an improved clinical stage and shorter overall survival [2,9,34]. For cellular activities, PRR11 was involved in proliferation, migration, cell-cycle progression, invasion, apoptosis and autophagy of NSCLC cells [2,9,10]. Similarly, in vivo tumorigenicity could also be modulated by PRR11 [2,9]. Functionally, PRR11 activated phosphorylation of Akt and mTOR and recruited the actin-related protein 2/3 complex, thus activating Akt/mTOR signaling and F-actin assembly [10,35]. Moreover, a complex, regulated by p53, consisting of PRR11 and SKA2 contributed to the progression of NSCLC cells [3,36]. As for the transcription level, PRR11 targeted the E2F1/pituitary tumor-transforming gene 1 (PTTG1) axis with involvement of NSCLC progression [16]. Preliminary studies on treating NSCLC patients demonstrated that lncRNA DLX6-AS1 knockdown inhibited cell proliferation, migration, invasion and initiated apoptosis targeting PRR11 in NSCLCs [37]. Collectively, PRR11 could be a reliable prognostic factor and therapeutic target for NSCLC patients.

Breast Cancer (BRCA)
BRCA, the most common solid tumor among women, is the second cause of tumorrelated death in women [38]. BRCA has four categories, including estrogen receptor (ER + ), progesterone receptor (PR + ), human epidermal receptor 2 (HER2 + ) and triple negative breast cancer (TNBC), which correlate to the expression of hormonal receptors [39]. However, the progress in early diagnosis and treatment for BRCA patients is still far from satisfactory, appealing to more reliable biomarkers. In 109 BRCA patients and 260 ER + BRCA patients, PRR11 promotes more lymph node metastasis, higher Ki-67 ratio and endocrine resistance, thus diminishing the overall survival, recurrence-free survival and relapse-free survival [1,5,40]. In cellular activities, PRR11 facilitated cellular proliferation, migration, invasion and antiestrogen resistance [1,5,17,18]. Subsequent functional investigation uncovered that PRR11 reduced the expression of E-cadherin, cytokeratin-18 and upregulated the expression of N-cadherin, vimentin and fibronectin targeting EMTinducing transcription factors Snail, Slug, zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2 [5]. Moreover, PRR11 blocked p85 homodimerization and sensitized to ligandinduced PI3K activation, suggesting that PRR11 amplification confers resistance to estrogen deprivation through hyperactivation of the PI3K pathway [1]. Notably, ultrasonic irradiation and SonoVue microbubbles-mediated RNA interference targeting PRR11 could exert anti-cancer effects via PRR11 [41]. However, the oncogenic role of PRR11 in vivo is still blurred, demanding additional exploration. In brief, PRR11 acts as a prognostic factor for BRCA patients, which could also be a reliable therapeutic target.

Gastric Cancer (GC) and Colorectal Cancer (CRC)
GC, originating from the epithelium of the gastric mucosa, evidently has regional differences [42]. Disappointingly, the early diagnosis rate of GC patients is still negligible, contributing to more distant metastasis and lymph node metastasis [43]. Accordingly, effective diagnostic indicators are pivotal to predict the outcomes of GC patients and facilitate the development of targeted therapy. In 216 GC patients, PRR11 expression is positively correlated with TNM stage and tumor differentiation, accompanied by the shorter overall survival of these patients. Further exploration on cellular activities illustrated that PRR11 overexpression promoted cellular proliferation and colony formation of GC cells [13]. As the vital cell subsets in tumorigenesis, GC stem cells were also modulated by PRR11 in self-renewal and maintaining stemness [6]. Moreover, in vivo tumorigenicity was suppressed by PRR11 in tumor volume and weight. Functionally, PRR11 exerted oncogenic effects mainly upregulating collagen triple helix repeat containing 1 (CTHRC1), downregulating latexin (LXN) or targeting MAPK signaling [6,13].
Nowadays, CRC is the fourth most deadly cancer, with low diagnostic rates and approximately 900,000 deaths annually [44]. Though organoids could provide long-term cultures of normal intestinal epithelial cells and good animal models for CRC, the prognosis of CRC patients is still poor [45]. Compared to normal ones, PRR11 is obviously elevated in CRC tissues and cells. Exploration of its biological activities showed that PRR11 silencing suppressed the proliferation, invasion and migration of CRC cells and xenograft tumor growth in vivo. Mechanically, PRR11 knockdown suppressed the EGFR/ERK/AKT pathway via inhibiting CTHRC1 expression [19]. However, further survival analysis of CRC patients targeting PRR11 expression is still in urgent demand. In summary, PRR11 might have an oncogenic role in the progression and development of GC and CRC patients.

Hepatocellular Carcinoma (HCC) and Hilar Cholangiocarcinoma (HCCA)
HCC, characterized with high morbidity, is the most common primary liver cancer in humans [46]. Though surgical resection and adjuvant chemotherapy of sorafenib would improve overall survival of HCC patients, the incidence of HCC and the mortality of HCC patients were still high [47,48]. Due to the lack of explicit clinical symptoms in the early stage, effective diagnostic biomarkers are in urgent need. Analysis of the weighted gene co-expression network in HCC found that PRR11 is positively connected to the progression and prognosis of HCC patients [49]. Further exploration on clinical characteristics of 80 pairs of HCC tissues and adjacent non-tumor liver tissues confirmed that PRR11 overexpression contributes to larger tumor size, improved TNM stage and shorter overall survival. In cellular activities, PRR11 silencing inhibited cellular proliferation, migration and invasion in vitro. Similar inhibitory effects on xenograft tumor growth of HCC cells were also observed in tumor volume, weight and Ki-67 ratio. Functionally, PRRl1 knockdown reduced the expression of N-cadherin while increasing E-cadherin expression, thus inactivating the EMT process. Furthermore, PRR11 was positively correlated with β-catenin, c-myc and cyclin D1 targeting β-catenin signaling [20]. Of course, PRR11 targeted the E2F1/PTTG1 axis to modulate gene transcription of HCC cells [16].
HCCA, also known as Klatskin tumor, is a common malignant tumor of the biliary system [50]. Despite current advances in imaging and surgical techniques, the prognosis of HCCA patients is still poor due to its special location, aggressive growth and adjacent relationship with the hilar [51]. In order to facilitate early diagnosis, novel biomarkers should be identified for HCCA. In 49 patients with HCCAs, PRR11 overexpression is linked to earlier invasion, more lymph node metastasis and shorter overall survival. For cellular activities, PRR11 knockdown suppressed hilar cholangiocarcinoma cell proliferation, migration, cell-cycle progression and tumor growth in vitro and in vivo. Subsequent functional analysis illustrated that PRR11 increased the expression of vimentin (VIM) protein and decreased expression of E-cadherin, thus initiating the EMT process. There were also some other downstream regulatory proteins, including ubiquitin carboxyl-terminal hydrolase 1 (UCHL1), early growth response protein (EGR1) and system a amino acid transporter 1 (SNAT1) [12]. There is also a demand for additional experiments on cellular apoptosis and autophagy in HCC and HCCA cells. Consequently, PRR11 plays an oncogenic role in the progression of HCC and HCCA, which might be a valuable prognostic marker and therapeutic target.

Pancreatic Cancer
Pancreatic cancer, which exhibits extensive metabolic reprogramming, is one of the malignant tumors of the digestive tract [52]. The clinical features of pancreatic cancer, including late diagnosis, early metastasis and limited reaction to chemotherapy or radiotherapy, lead to a poor five-year survival rate, which is around 10% [53]. To improve early diagnosis and overall survival, new biomarkers should be further explored. The expression level of PRR11 in patients with pancreatic cancers was significantly higher than those of normal patients [14]. Analysis of clinical parameters of 38 pancreatic cancer samples and 10 normal pancreatic tissues demonstrated that PRR11 overexpression is positively correlated with tumor invasion and differentiation, accompanied by shorter overall survival [14,54]. In vitro, PRR11 knockdown markedly inhibited cellular migration in pancreatic cancer cells [14]. Mechanically, the E2F1/PTTG1 axis was regulated by PRR11 to participate in the progression of PAAD cells [16]. Notably, further exploration of treating pancreatic cancers revealed that ubiquitin specific protease 34 (USP34) knockdown inhibited proliferation and migration, and induced apoptosis targeting PRR11 [55]. Moreover, miR-144-3p decreased PRR11 expression to oppress cell proliferation and initiate cell apoptosis and cell cycle arrest [56]. In summary, PRR11 might be a reliable prognostic factor for pancreatic cancer patients, for which the biological interventions should be further validated.

Ovarian Cancer
Ovarian cancer, the third most common gynecologic malignancy, mainly caused deaths in patients with gynecological tumors [57]. Despite the progress in comprehensive treatment, 60-70% of ovarian cancer patients are usually in the advanced clinical stage, leading to poor prognosis [58,59]. Hence, reliable indicators for diagnosis and treatment of ovarian cancer patients are indispensable. Compared to normal ovarian surface epithelium tissues and a normal ovarian epithelial cell line IOSE80, PRR11 expression is significantly upregulated in 51 pairs of ovarian cancer tissues and 4 ovarian cancer cell lines, including Caov3, SKOV3, OVCAR3 and HO-8910 [7]. Concluding from the clinical features of 51 pairs of ovarian cancer patients and 49 primary invasive ovarian cancer patients, PRR11 is positively correlated with improved FIGO stage, larger tumor size, more lymph node metastasis and shorter overall survival [7,11]. For cellular activities, cell proliferation, migration and invasion were involved in the biological functions of PRR11 [7]. In addition, PRR11 obviously promoted xenograft tumor growth in vivo [11]. Mechanically, PRR11 targeted c-myc, cyclin D1, matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteases 2 (TIMP-2), EGR1 and N-cadherin to exert the oncogenic effects on cell proliferation and migration [7,11]. PRR11 also increased the expression of p-Akt/Akt and cytoplasmic and nuclear β-catenin, thus activating PI3K/Akt/β-catenin signaling [7]. However, there is still a demand for the investigation of PRR11 activities on cell cycle, apoptosis and autophagy. To summarize, PRR11 could be a reliable prognostic factor for ovarian cancer patients.

Osteosarcoma
Osteosarcoma is the most prevalent and malignant bone cancer in children and adolescents [60]. With dramatic improvement in chemotherapy and surgery, the long-term survival rates of osteosarcoma patients are only 70% due to its variable distribution, distant metastases and lack of clinical symptoms [61]. Therefore, reliable biomarkers for osteosarcoma patients are in urgent demand. Compared with paracancerous tissues, PRR11 expression is elevated in 62 cases of osteosarcoma tissues. In further analysis of PRR11 expression in osteosarcoma patients, high PRR11 expression is correlated with larger tumor size, advanced Enneking stage and lymph node metastasis. Relative higher expression of PRR11 was also observed in osteosarcoma cell lines, including SAOS2, MG63 and U2OS. In cellular activities, PRR11 was involved in affecting proliferation, migration, invasion and apoptosis. Mechanically, PRR11 knockdown decreased β-catenin and p-GSK-3β and increased p-β-catenin and GSK-3β, thus inactivating Wnt/β-catenin signaling. In addition, PRR11 knockdown also increased the expression of E-cadherin, but reduced vimentin and fibronectin in the EMT signaling [21]. Above all, PRR11 might be an independent and reliable biomarker and therapeutic target for osteosarcoma patients.

PRR11 Involved Targeted Therapies in Various Malignancies
In recent years, targeted therapies have gradually exhibited potential application in cancer treatment with extraordinary clinical efficacy [67,68]. Based on these investigational results, targeted therapies of PRR11, especially interactions between PRR11 and miRNAs, lncRNAs and other molecules, would be utilized for cancer treatment.

Interactions between PRR11 and miRNAs
The miRNAs, a group of non-coding RNAs, play vital roles in the initiation and progression of human cancers [69]. In most circumstances, miRNAs induce the degradation and translational repression of target mRNAs to modulate gene expression targeting the 3 untranslated region (3 UTR) of mRNAs [70]. For instance, the luciferase activity of the PRR11 3-UTR wild-type reporter gene, not the PRR11 3-UTR mutated reporter gene, was significantly decreased by miRNA-26b-5p, thus suppressing PRR11 expression [71]. Similarly, in prostate cancer, osteosarcoma, pancreatic cancer and breast cancer, miR-195, miRNA-211-5p, miR-204-5p and miR-144-3p directly targeted PRR11 3 -UTR to participate in cellular activities, including proliferation, migration, cell cycle, apoptosis and angiogenesis [18,56,72,73]. Therefore, exploring more miRNAs targeting PRR11 3 -UTR might be a reliable approach to regulate PRR11 expression in cancer treatment.

Interactions between PRR11 and other Molecules
Preliminary experiments found that SonoVue microbubbles-mediated RNA interference targeting PRR11 could exert anti-cancer effects, forecasting the potential application of PRR11 for targeted therapy [9,41]. In the meanwhile, circRNA_cZNF292 was involved in angiogenesis of glioma through PRR11 [77]. Notably, some biological molecules were also involved in PRR11-related targeted therapies. USP34 facilitated pancreatic cancer cell progression targeting PRR11 [55]. Surprisingly, PRR11 also acted as a significant part in a limited number of clinical cases. Chalcone, modificated by dithiocarbamate scaffolds, exert inhibitory effects via PRR11 downregulation [30]. Furthermore, PRR11 was involved in the treatment of ultrasonic irradiation for breast cancer [41]. Therefore, targeting PRR11 might provide a reliable and promising therapy for human malignancies (Figure 3).

Conclusions and Future Prospective
Concluding from the experimental and clinical data reviewed above, PRR11 is involved in cellular activities via several signaling pathways and biological molecules, manifesting its potential application in the targeted therapies. As for its contribution in tumorigenicity, PRR11 facilitates cellular proliferation in vitro in BRCA, Pancreatic cancer and Osteosarcoma; likewise, it contributes to the tumorigenicity of in vitro and in vivo models in TSCC, ESCC, NSCLC, GC, CRC, HCC, HCCA and Ovarian cancer. In considering clinical characteristics of patients with various solid tumors, PRR11 is more or less significantly correlated with greater tumor size, higher Ki-67, advanced clinical stage, more lymph node metastasis, more tumor differentiation and higher recurrence. Hence, PRR11 serves as a reliable prognostic and diagnostic indicator in TSCC, NSCLC, BRCA, GC, HCC, HCCA, Pancreatic cancer and Ovarian cancer. Moreover, traumatic autophagy, instead of protective autophagy, is notably reduced by PRR11 in cancer cells. Though PRR11 expression is downregulated in LAML patients from the GEO database, the oncogenic role of PRR11 in a variety of human cancers have already been verified. Therefore, PRR11 exerts oncogenic effects on several human cancers, which could be applied into clinical cancer treatment.
However, the existing research data of PRR11 is still not integrated. For instance, the clinical significance and cellular activities of the human cancers mentioned above are more or less in deficiency, thus clinical application would encounter great difficulties. Moreover, in gastric cancer stem cells, Hu et al. firstly applied dorsomorphin, one of the

Conclusions and Future Prospective
Concluding from the experimental and clinical data reviewed above, PRR11 is involved in cellular activities via several signaling pathways and biological molecules, manifesting its potential application in the targeted therapies. As for its contribution in tumorigenicity, PRR11 facilitates cellular proliferation in vitro in BRCA, Pancreatic cancer and Osteosarcoma; likewise, it contributes to the tumorigenicity of in vitro and in vivo models in TSCC, ESCC, NSCLC, GC, CRC, HCC, HCCA and Ovarian cancer. In considering clinical characteristics of patients with various solid tumors, PRR11 is more or less significantly correlated with greater tumor size, higher Ki-67, advanced clinical stage, more lymph node metastasis, more tumor differentiation and higher recurrence. Hence, PRR11 serves as a reliable prognostic and diagnostic indicator in TSCC, NSCLC, BRCA, GC, HCC, HCCA, Pancreatic cancer and Ovarian cancer. Moreover, traumatic autophagy, instead of protective autophagy, is notably reduced by PRR11 in cancer cells. Though PRR11 expression is downregulated in LAML patients from the GEO database, the oncogenic role of PRR11 in a variety of human cancers have already been verified. Therefore, PRR11 exerts oncogenic effects on several human cancers, which could be applied into clinical cancer treatment.
However, the existing research data of PRR11 is still not integrated. For instance, the clinical significance and cellular activities of the human cancers mentioned above are more or less in deficiency, thus clinical application would encounter great difficulties. Moreover, in gastric cancer stem cells, Hu et al. firstly applied dorsomorphin, one of the specific inhibitors of MAPK signaling, to just simply explore its involvement with PRR11 activities [6]. Conversely, our reviewed results focus on a limited number of human cancers, appealing to the reliable evaluation for PRR11 expression in other tumors. Univariate Cox regression analysis of the cancer genome atlas (TCGA) and GEO databases might be an option [78]. Additionally, the significance of clinical samples is testified [29]. Gu et al. also verified the feasibility of weighted gene co-expression network analysis (WGCNA) [49]. Hence, there is an urgent demand to refine the current data and probe the role of PRR11 in other tumors.
Of course, there are still some preliminary explorations in the correlation of PRR11 and upstream or downstream targets, further promoting its application in targeted therapy. The MirTarget program can discern the number of miRNAs binding sites with the genes [79]. The technology of the protein chip system could also be utilized for screening upstream or downstream targets of PRR11 [80]. Furthermore, functional gain or loss experiments might be reliable options [1,10].
All the above findings reveal that PRR11 serves as an oncogenic factor in cellular proliferation, migration, invasion, cell-cycle progression, apoptosis and autophagy in human cancers, including TSCC, ESCC, NSCLC, BRCA, GC, CRC, HCC, HCCA, Pancreatic cancer, Ovarian cancer and Osteosarcoma. Moreover, PRR11 positively correlates with clinical stage, tumor size, lymph node metastasis and tumor differentiation, leading to poor overall survival. Further mechanical investigation demonstrates that EMT, PI3K/AKT/mTOR, Wnt/β-catenin signalings and biological molecules, including MMP2, TIMP-2, p21, p27, CDK2, cyclin A2, CTHRC1, LXN, EGR1, UCHL1, SNAT1 and PTTG1, are involved in the functional process of PRR11, providing potential clinical applications of PRR11 for targeted therapies. While the existing data is still far from satisfactory, further investigation should be performed for the verification of the oncogenic role of PRR11.

Conflicts of Interest:
The authors declare no conflict of interest.