The pluripotency factor LIN28B is involved in oral carcinogenesis and associates with tumor aggressiveness and unfavorable prognosis

Oral squamous cell carcinoma (OSCC) is one of the common solid cancers worldwide with well-known etiologic factors including smoking abuse, alcohol consumption as well as HPV infection, etc. [1]. Despite significant progress in the combined and sequential treatment for OSCC in the last decades, however, the long-term survival rate has not been remarkably improved [2]. Much efforts have been made to unravel the genetic and epigenetic abnormalities behind OSCC pathogenesis and enable us to capture the mechanistic insights into oral cancer progression [3]. However, until now, few biomarkers have been unequivocally established for diagnostic and prognostic management of oral cancer. This unmet challenge significantly impedes the improvement of patients’ treatment outcomes [4]. Therefore, the novel biomarkers discovery and validation is urgent and of great importance for early detection, novel therapeutics development, ultimately leading to improved prognosis.

LIN28B and its mammalian homologue LIN28 are two RNA-binding protein emerged as key modulators for stem cell maintenance, cellular metabolism as well as tumorigenesis [5, 6]. LIN28B and LIN28, initially identified as key regulators of developmental timing in Caenorhabditis elegans, were known as master cell reprogramming factors to induce pluripotency in conjunction with KLF4, OCT4 and Sox2 [7, 8]. Mounting evidence has revealed that LIN28B/LIN28 functions as negative regulators of microRNA biogenesis, selectively represses let-7 family production in diverse physiological and pathological settings, although they act in different ways [9]. Moreover, they have been implicated in tumorigenesis and usually aberrantly overexpressed in multiple human cancers including hepatocellular, colon, intestinal and head neck cancers. Their overexpression commonly associated with tumor aggressive behaviors, advanced clinicopathological features, and decreased survival [10‐13]. Gain-and loss-of-function studies have further revealed that LIN28B/LIN28 promotes cancer cell proliferation, invasion and metastasis both in vitro and in vivo largely through let-7 repression [10, 12, 14]. Noticeably, comprehensive analyses of LIN28 and LIN28B expression in human cancers pointed to LIN28B as the more relevant homologue underlying tumorigenesis [15]. Further evidence linking LIN28B to tumorigenesis derived from several previous findings that it facilitated cellular malignant transformation in vitro [14‐16] and transgenic overexpression was sufficient to initiate neuroblastoma and hepatoblastoma and also required for their maintenance in murine models [11, 17]. Moreover, conditional LIN28B deletion or siRNA-mediated LIN28B knockdown reduced tumor burden, inhibited metastasis and prolonged survival in vivo [10, 18]. Collectively, these findings have provided strong evidence that LIN28B is a bona fide oncogene mediating cancer initiation and progression, and also a novel therapeutic target against cancer.

Accumulating evidence has revealed essential clues regarding LIN28B expression and potential roles in oral cancers [19, 20]. We have revealed a functional single nucleotide polymorphism [variant allele (T) of rs221636] in LIN28B,which is significantly associated with oral cancer susceptibility in a Chinese population, thus supporting the key roles of LIN28B during oral tumorigenesis [21]. However, the expression pattern of LIN28B and its clinicopathological significance in OSCC have not been definitively established yet. In the present study, we sought to determine the LIN28B expression in OSCC cell line, animal model and primary human OSCC specimens, and further identify potential relationships between its abundance and clinicopathological features as well as patients’ prognosis.

Accumulating evidence has revealed that the pluripotency factor LIN28B, a highly conserved RNA-binding protein, has emerged as a key player in many developmental and pathological processes including stem cell hemostasis, cellular metabolism and tumorigenesis [15, 28, 29]. Of great interests, it has been found to mediate cellular malignant transformation, fuel cancer cell proliferation, invasion and metastasis cascade as well as promote cancer stem cell maintenance in diverse human cancers [6, 10, 14]. Its overexpression significantly associated with cancer aggressiveness and poor prognosis [14, 15]. These findings indicate that LIN28B is an bona fide oncogene that might be exploited as cancer biomarker and therapeutic target. Here, we measured the expression patterns of LIN28B in OSCC cells, chemical-induced OSCC animal model and primary OSCC samples, and further determined its clinicopathological significance. Our findings reveal that LIN28B might be involved in OSCC initiation and progression by serving as an oncogene. Its overexpression associates with aggressive clinicopathological features and unfavorable patients’ prognosis in a significant fraction of OSCC.

The human oral tumorigenesis is characterized by multiple and consecutive histopathological stages from normal epithelial to invasive SCC driven by oncogenes activation and tumor suppressor inactivation [30]. This has been elegantly reproduced in chemical-induced animal models, such as DMBA-induced buccal SCC in hamster and 4-nitroquinoline 1-oxide (4NQO)-induced oral SCC in rodents [23, 24, 31]. In the present study, we utilized the former model and determined LIN28B expression during OSCC tumorigenesis by immunohistochemistry. As anticipated, our findings established that LIN28B expression was almost negative in normal epithelial, but its positive staining gradually became more and more in dysplasia and SCC samples, suggesting that LIN28B served as an oncogene critically involved in OSCC pathogenesis. This is well consistent with the oncogenic roles of LIN28B driving cancer initiation [11]. Because a line of evidence has revealed that LIN28B alone is capable to induce cell transformation in vitro and sufficient to initiate multiple cancers in vivo [11, 15‐17]. Collectively, our data provide the in vivo evidence to support the oncogenic roles of LIN28B in oral tumorigenesis. Of course, further in-depth investigations into the detailed mechanism regarding LIN28B behind OSCC are warranted in future.

Previous findings have indicated that LIN28B is usually overexpressed in a broad spectrum of human cancers and is critically required for cancer overgrowth [15, 18]. Importantly, overexpression of LIN28B significantly associated with advanced stages, lymph node metastasis and unfavorable prognosis in diverse cancers including oral cancer [18, 20]. For example, Wu and his colleague reported that LIN28 and LIN28B expression levels were both increased in 72 primary OSCC and positively correlated with gender, tumor differentiation and patients’ survival [20]. Consistently, our data further extended these previous findings and determined LIN28B abundance in a panel of OSCC cell lines and an independent cohort of primary OSCC. Our results revealed that Lin28B was abnormally overexpressed in OSCC cells and human samples. Noticeably, elevated LIN28B significantly associated with tumor size and advanced clinical stages, but not the gender and tumor differentiation status as reported by Wu [20]. We reasoned that this discrepancy might be due to inherent tumor heterogeneity and different immunohistochemical scores and patient stratification. Interestingly, when the manuscript was in preparation, Lin and his colleague reported that elevated LIN28B correlated with lymph node metastasis in OSCC, although the mRNA abundance of LIN28B was utilized as readout by quantitative real-time PCR assay in 20 paired samples [32]. Therefore, our findings together with others strongly support the notion that LIN28B is aberrantly overexpressed in OSCC and significantly correlated with malignant clinicopathological features.

Although past decades have witnessed much progress in diagnosis and treatment for OSCC, however, the long-term survival still remains disappointing. Accurate prognostic prediction regime for OSCC is a great challenge, but highly beneficial in the clinic. Evidence has suggested that LIN28B may function as a key prognostic predictor for patients with colon, oesophagus cancer and neuroblastoma [17, 18, 33]. Indeed, our results indicated that high LIN28B correlated with decreased patients’ survival and served as an independent prognostic factor affecting patients’ survival. This finding is in agreement with previous report where they showed that patients with high LIN28B had earlier recurrence and decreased overall survival rates [20]. Thus, these data indicate that the expression status of LIN28B might serve as not only a novel diagnostic, but also a prognostic biomarker for OSCC. However, a large cohort of patients with long-term follow-up is still needed to definitively establish the prognostic values of LIN28B for OSCC.

In conclusion, our findings reveal that LIN28B might be critically involved in OSCC initiation and progression. Its upregulation correlates with aggressive clinicopathological parameters and adverse prognosis, thus suggesting that LIN28B might serve as a novel cancer biomarker as well as therapeutic target for OSCC.