Shi-Long Lu

CIP2A Inhibits PP2A in Human Malignancies

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, we describe a cellular inhibitor of PP2A with oncogenic activity. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, our data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies.

Distinct mechanisms of TGF-β1–mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis

In the present study, we demonstrated that human skin cancers frequently overexpress TGF-beta1 but exhibit decreased expression of the TGF-beta type II receptor (TGF-(beta)RII). To understand how this combination affects cancer prognosis, we generated a transgenic mouse model that allowed inducible expression of TGF-beta(1) in keratinocytes expressing a dominant negative TGF-(beta)RII (Delta(beta)RII) in the epidermis. Without Delta(beta)RII expression, TGF-beta1 transgene induction in late-stage, chemically induced papillomas failed to inhibit tumor growth but increased metastasis and epithelial-to-mesenchymal transition (EMT), i.e., formation of spindle cell carcinomas. Interestingly, Delta(beta)RII expression abrogated TGF-beta1-mediated EMT and was accompanied by restoration of membrane-associated E-cadherin/catenin complex in TGF-beta1/Delta(beta)RII compound tumors. Furthermore, expression of molecules thought to mediate TGF-beta1-induced EMT was attenuated in TGF-beta1/Delta(beta)RII-transgenic tumors. However, TGF-beta1/Delta(beta)RII-transgenic tumors progressed to metastasis without losing expression of the membrane-associated E-cadherin/catenin complex and at a rate higher than those observed in nontransgenic, TGF-beta1-transgenic, or Delta(beta)RII-transgenic mice. Abrogation of Smad activation by Delta(beta)RII correlated with the blockade of EMT. However, Delta(beta)RII did not alter TGF-beta1-mediated expression of RhoA/Rac and MAPK, which contributed to increased metastasis. Our study provides evidence that TGF-beta1 induces EMT and invasion via distinct mechanisms. TGF-beta1-mediated EMT requires functional TGF-(beta)RII, whereas TGF-beta1-mediated tumor invasion cooperates with reduced TGF-(beta)RII signaling in tumor epithelia.

Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression

TGF-beta and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4-/- mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3-/- mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2-/- tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2-/- mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

Overexpression of transforming growth factor β1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation

In the present study, we show that transforming growth factor β1 (TGF-β1) was frequently overexpressed in human head and neck squamous cell carcinomas (HNSCCs) and adjacent tissues in comparison with normal head and neck tissues. To determine the role of TGF-β1 overexpression in HNSCC carcinogenesis, we generated transgenic mice in which TGF-β1 transgene expression can be induced in head and neck epithelia. TGF-β1 transgene induction in head and neck epithelia, at levels similar to those in human HNSCCs, caused severe inflammation and angiogenesis. Consequently, TGF-β1-transgenic epithelia exhibited hyperproliferation. These phenotypes correlated with enhanced Smad signaling in transgenic epithelia and stroma. Our study suggests that TGF-β1 overexpression at early stages of HNSCC formation provides a tumor promoting microenvironment.

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation

Smad4 is a central mediator of TGF-beta signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4-/- mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited severe inflammation, which was associated with increased expression of TGF-beta1 and activated Smad3. We present what we believe to be the first single gene-knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.

Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis.

It has been shown that Smad3 exerts both tumor-suppressive and -promoting roles. To evaluate the role of Smad3 in skin carcinogenesis in vivo, we applied a chemical skin carcinogenesis protocol to Smad3 knockout mice (Smad3−/− and Smad3+/−) and wild-type littermates (Smad3+/+). Smad3−/− mice exhibited reduced papilloma formation in comparison with Smad3+/+ mice and did not develop any squamous cell carcinomas. Further analysis revealed that Smad3 knockout mice were resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced epidermal hyperproliferation. Concurrently, increased apoptosis was observed in TPA-treated Smad3−/− skin and papillomas when compared with those of wild-type mice. Expression levels of activator protein-1 family members (c-jun, junB, junD, and c-fos) and transforming growth factor (TGF)-α were significantly lower in TPA-treated Smad3−/− skin, cultured keratinocytes, and papillomas, as compared with Smad3+/+ controls. Smad3−/− papillomas also exhibited reduced leukocyte infiltration, particularly a reduction of tumor-associated macrophage infiltration, in comparison with Smad3+/+ papillomas. All of these molecular and cellular alterations also occurred to a lesser extent in Smad3+/− mice as compared with Smad3+/+ mice, suggesting a Smad3 gene dosage effect. Given that TGF-β1 is a well-documented TPA-responsive gene and also has a potent chemotactic effect on macrophages, our study suggests that Smad3 may be required for TPA-mediated tumor promotion through inducing TGF-β1–responsive genes, which are required for tumor promotion, and through mediating TGF-β1–induced macrophage infiltration.

Role of TGFβ in skin inflammation and carcinogenesis

The functions of transforming growth factor β‐1(TGFβ1) are cell‐context specific. We have found that TGFβ1 expression in human skin squamous cell carcinoma (SCC) samples has two distinct distribution patterns: (1) either predominantly in suprabasal layers or (2) throughout tumor epithelia including basal proliferative cells. To understand whether the spatial TGFβ1 expression patterns affect its functions, we have generated several keratinocyte‐specific transgenic mouse models in which TGFβ1 overexpression can be induced either predominantly in the suprabasal epidermis or in the basal layer of the epidermis and hair follicles. Suprabasal TGFβ1 overexpression inhibits keratinocyte proliferation, suppresses skin carcinogenesis at early stages, but promotes tumor invasion at later stages. In contrast, TGFβ1 overexpression in the basal layer of the epidermis and hair follicles causes a severe inflammatory skin disorder and epidermal hyperproliferation. Given the importance of inflammation in cancer development, our data suggest that TGFβ1‐induced skin inflammation may override its tumor suppressive effect at early stages during skin carcinogenesis. This hypothesis is further suggested by our recent study that Smad3 knockout mice are resistant to skin chemical carcinogenesis at least in part via abrogation of endogenous TGFβ1‐induced inflammation. This review intends to summarize current insights into the role of TGFβ1 in skin inflammation and carcinogenesis.

Methylation of microRNA-9 is a specific and sensitive biomarker for oral and oropharyngeal squamous cell carcinomas

Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.

Reactivation of Developmentally Expressed p63 Isoforms Predisposes to Tumor Development and Progression

Genes that are active during normal development are frequently reactivated during neoplastic transformation. We now report that developmentally expressed TAp63 isoforms are frequently reactivated in human squamous cell carcinomas. To determine the consequences of TAp63 reactivation, we induced TAp63alpha expression during chemically-induced skin carcinogenesis. Deregulated TAp63alpha expression dramatically accelerated tumor development and progression, frequently resulting in epithelial-mesenchymal transitions to spindle cell carcinomas and lung metastases. Consistent with this observation, we detected high levels of Twist and N-cadherin in tumors overexpressing TAp63alpha. Thus, as observed for other developmental pathways, aberrant reactivation of TAp63 predisposes to tumor development and progression.

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.