βII‐Spectrin (SPTBN1) is an adapter protein for Smad3/Smad4 complex formation during transforming growth factor beta (TGF‐β) signal transduction. Forty percent of SPTBN1^+/− mice spontaneously develop hepatocellular carcinoma (HCC), and most cases of human HCC have significant reductions in SPTBN1 expression. In this study, we investigated the possible mechanisms by which loss of SPTBN1 may contribute to tumorigenesis. Livers of SPTBN1^+/− mice, compared to wild‐type mouse livers, display a significant increase in epithelial cell adhesion molecule‐positive (EpCAM⁺) cells and overall EpCAM expression. Inhibition of SPTBN1 in human HCC cell lines increased the expression of stem cell markers EpCAM, Claudin7, and Oct4, as well as decreased E‐cadherin expression and increased expression of vimentin and c‐Myc, suggesting reversion of these cells to a less differentiated state. HCC cells with decreased SPTBN1 also demonstrate increased sphere formation, xenograft tumor development, and invasion. Here we investigate possible mechanisms by which SPTBN1 may influence the stem cell traits and aggressive behavior of HCC cell lines. We found that HCC cells with decreased SPTBN1 express much less of the Wnt inhibitor kallistatin and exhibit decreased β‐catenin phosphorylation and increased β‐catenin nuclear localization, indicating Wnt signaling activation. Restoration of kallistatin expression in these cells reversed the observed Wnt activation. Conclusion: SPTBN1 expression in human HCC tissues is positively correlated with E‐cadherin and kallistatin levels, and decreased SPTBN1 and kallistatin gene expression is associated with decreased relapse‐free survival. Our data suggest that loss of SPTBN1 activates Wnt signaling, which promotes acquisition of stem cell‐like features, and ultimately contributes to malignant tumor progression. (Hepatology 2015;61:598‐612)

The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In this study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathologic changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to 5 months. Histopathologic changes occurred concurrently with an increase in an inflammatory, invasive, metabolic, and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning 1 week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTOR signaling that were attenuated by treatment with the mTOR inhibitor everolimus. Our findings are the first to show a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. Cancer Res; 73(14); 4349–61. ©2013 AACR.

MLL2, an epigenetic regulator in mammalian cells, mediates histone 3 lysine 4 tri-methylation (H3K4me3) through the formation of a multiprotein complex. MLL2 shares a high degree of structural similarity with MLL, which is frequently disrupted in leukemias via chromosomal translocations. However, this structural similarity is not accompanied by functional equivalence. In light of this difference, and previous reports on involvement of epigenetic regulators in malignancies, we investigated MLL2 expression in established cell lines from breast and colon tissues. We then investigated MLL2 in solid tumors of breast and colon by immunohistochemistry, and evaluated potential associations with established clinicopathologic variables.

We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. We demonstrated that PEITC inhibits the growth of prostate cancer cells with different “hotspot” p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. Our studies provide the first evidence that PEITC’s anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.

Recent studies report that the polarity gene myelin and lymphocyte protein 2 (MAL2), is overexpressed in multiple human carcinomas largely at the transcript level. Because chromosome 8q24 amplification (where MAL2 resides) is associated with hepatocellular- and cholangio-carcinomas, we examined MAL2 protein expression in these human carcinoma lesions and adjacent benign tissue using immunohistochemistry. For comparison, we analyzed renal cell carcinomas that are not associated with chromosome 8q24 amplification. Surprisingly, we found that MAL2 protein levels were decreased in the malignant tissues compared to benign in all three carcinomas, suggesting MAL2 expression may be anti-oncogenic. Consistent with this conclusion, we determined that endogenously overexpressed MAL2 in HCC-derived Hep3B cells or exogenously expressed MAL2 in hepatoma-derived Clone 9 cells (that lack endogenous MAL2) promoted actin-based protrusion formation with a reciprocal decrease in invadopodia. MAL2 overexpression also led to decreased cell migration, invasion and proliferation (to a more modest extent) while loss of MAL2 expression reversed the phenotypes. Mutational analysis revealed that a putative Ena/VASP homology 1 recognition site confers the MAL2-phenotype suggesting its role in tumor suppression involves actin remodeling. To reconcile decreased MAL2 protein expression in human carcinomas and its anti-oncogenic phenotypes with increased transcript levels, we propose a transcriptional regulatory model for MAL2 transient overexpression.