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Supplementary Data from Sox7 Is an Independent Checkpoint for β-Catenin Function in Prostate and Colon Epithelial Cells

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins including p27kip1, p21cip1, p57kip2, p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway. (Mol Cancer Res 2009;7(1):12–22)

The presence of somatic β-catenin mutations in some prostate cancers implies that aberrant WNT signaling is involved in the cancer development. Although β-catenin stability is regulated by a multicomponent destruction complex, mutational alterations of β-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, β-catenin may be regulated by another protein in the prostate. In fact, recent linkage and somatic deletion analyses in prostate cancers reveal a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene. Here we show that Sox7 protein expression was indeed down-regulated in 47% (15 of 32) of prostate adenocarcinomas. In addition, Sox7 mRNA was down-regulated in 60% of snap-frozen tumors. This down-regulation was found to be due to tumor-specific promoter hypermethylation, which was present in 48% (10 of 21) of primary prostate tumors and 44% (11 of 25) of prostate cancer cell lines/xenografts. We discovered that Sox7 protein physically interacts with β-catenin and suppresses β-catenin–mediated transcription by depleting active β-catenin. Furthermore, in HCT116 colorectal cancer cell lines with Sox7 inactivation, ectopic Sox7 expression suppressed cell proliferation and inhibited transcription that was activated by an endogenous mutant β-catenin. Although nearly all colorectal cancers contain mutations in β-catenin or adenomatous polyposis coli/axin, epigenetic silencing of Sox7 was still observed. These data suggest that Sox7 is a tumor suppressor that functions as an independent checkpoint for β-catenin transcriptional activity. Inactivation of Sox7 could promote the development of a majority of colorectal tumors and approximately half of prostate tumors. (Mol Cancer Res 2008;6(9):1421–10)

Supplementary Data from Suppression of Anoikis by SKP2 Amplification and Overexpression Promotes Metastasis of Esophageal Squamous Cell Carcinoma

SummaryHypercapnia, elevation of the partial pressure of CO2in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), mammalian ortholog of zfh2, which mediates hypercapnic immune suppression inDrosophila, is expressed in mouse and human MØs. Deletion ofZfhx3in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung diseases.Graphical abstract

Angiogenesis is a hallmark of tumorigenesis, and hepatocellular carcinoma (HCC) is hypervascular and therefore very dependent on angiogenesis for tumor development and progression. Findings from previous studies suggest that in HCC cells, hypoxia-induced factor 1α (HIF1A) and zinc finger homeobox 3 (ZFHX3) transcription factors functionally interact in the regulation of genes in HCC cells. Here, we report that hypoxia increases the transcription of the ZFHX3 gene and enhances the binding of HIF1A to the ZFHX3 promoter in the HCC cell lines HepG2 and Huh-7. Moreover, ZFHX3, in turn, physically associated with and was functionally indispensable for HIF1A to exert its angiogenic activity, as indicated by in vitro migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) and microvessel formation in xenograft tumors of HCC cells. Mechanistically, ZFHX3 was required for HIF1A to transcriptionally activate the vascular endothelial growth factor A (VEGFA) gene by binding to its promoter. Functionally, down-regulation of ZFHX3 in HCC cells slowed their tumor growth, and addition of VEGFA to conditioned medium from ZFHX3-silenced HCC cells partially rescued the inhibitory effect of this medium on HUVEC tube formation. In human HCC, ZFHX3 expression was up-regulated, and this up-regulation correlated with both HIF1A up-regulation and worse patient survival, confirming a functional association between ZFHX3 and HIF1A in human HCC. We conclude that ZFHX3 is an angiogenic transcription factor that is integral to the HIF1A/VEGFA signaling axis in HCC cells.

Prostate development depends on balanced cell proliferation and differentiation, and acetylated KLF5 is known to alter epithelial proliferation. It remains elusive whether post-translational modifications of transcription factors can differentially determine adult stem/progenitor cell fate. Here we report that, in human and mouse prostates, Klf5 is expressed in both basal and luminal cells, with basal cells preferentially expressing acetylated Klf5. Functionally, Klf5 is indispensable for maintaining basal progenitors, their luminal differentiation, and the proliferation of their basal and luminal progenies. Acetylated Klf5 is also essential for basal progenitors’ maintenance and proper luminal differentiation, as deacetylation of Klf5 causes excess basal-to-luminal differentiation; attenuates androgen-mediated organoid organization; and retards postnatal prostate development. In basal progenitor-derived luminal cells, Klf5 deacetylation increases their proliferation and attenuates their survival and regeneration following castration and subsequent androgen restoration. Mechanistically, Klf5 deacetylation activates Notch signaling. Klf5 and its acetylation thus contribute to postnatal prostate development and regeneration by controlling basal progenitor cell fate., The role of the Klf5 in early and postnatal prostate development and in regeneration is unclear. Here, the authors show that Klf5 acetylation regulates and maintains luminal differentiation of prostate basal progenitors and is essential following androgen-induced regeneration.

Prostate cancer is the second leading cause of cancer-related death in the United States. As a first line treatment for hormone-refractory prostate cancer, docetaxel (DTX) treatment leads to suboptimal effect since almost all patients eventually develop DTX resistance. In this study, we investigated whether and how TGF-β affects DTX resistance of prostate cancer. Methods: Cytotoxicity of DTX in DU 145 and PC-3 cells was measured by CCK-8 and Matrigel colony formation assays. Resistance to DTX in DU 145 cells was examined in a xenograft tumorigenesis model. A luciferase reporter system was used to determine transcriptional activities. Gene expression was analyzed by RT-qPCR and Western blotting. Results: We found that KLF5 is indispensable in TGF-β-induced DTX resistance. Moreover, KLF5 acetylation at lysine 369 mediates DTX resistance in vitro and in vivo. We showed that the TGF-β/acetylated KLF5 signaling axis activates Bcl-2 expression transcriptionally. Furthermore, DTX-induced Bcl-2 degradation depends on a proteasome pathway, and TGF-β inhibits DTX-induced Bcl-2 ubiquitination. Conclusion: Our study demonstrated that the TGF-β-acetylated KLF5-Bcl-2 signaling axis mediates DTX resistance in prostate cancer and blockade of this pathway could provide clinical insights into chemoresistance of prostate cancer.

Simple Summary Prostate cancer is a leading cause of cancer-related deaths among men, and novel therapies for advanced PCa are urgently needed. Cardiac glycosides are a group of attractive candidates for anticancer repurposing, but deslanoside has not been tested for a potential anticancer effect so far. This study aims to test the anticancer effect of deslanoside in PCa and investigate the underlying mechanisms. Deslanoside effectively inhibited colony formation and tumor growth in multiple prostate cancer cell lines. Such an inhibitory effect involved both the cell cycle arrest at G2/M and the induction of apoptosis. Deslanoside altered the expression of many genes, which belonged to various cancer-associated cellular processes and signaling pathways. Altered expression levels for 15 deslanoside-modulated genes correlate with recurrence-free survival or overall survival in PCa patients, some of which have not been implicated in cancer before. Therefore, deslanoside exerts anticancer activity in PCa cells by modulating gene expression. Abstract Prostate cancer (PCa) is a leading cause of cancer-related deaths among men worldwide, and novel therapies for advanced PCa are urgently needed. Cardiac glycosides represent an attractive group of candidates for anticancer repurposing, but the cardiac glycoside deslanoside has not been tested for potential anticancer activity so far. We found that deslanoside effectively inhibited colony formation in vitro and tumor growth in nude mice of PCa cell lines 22Rv1, PC-3, and DU 145. Such an anticancer activity was mediated by both the cell cycle arrest at G2/M and the induction of apoptosis, as demonstrated by different functional assays and the expression status of regulatory proteins of cell cycle and apoptosis in cultured cells. Moreover, deslanoside suppressed the invasion and migration of PCa cell lines. Genome-wide expression profiling and bioinformatic analyses revealed that 130 genes were either upregulated or downregulated by deslanoside in both 22Rv1 and PC-3 cell lines. These genes enriched multiple cellular processes, such as response to steroid hormones, regulation of lipid metabolism, epithelial cell proliferation and its regulation, and negative regulation of cell migration. They also enriched multiple signaling pathways, such as necroptosis, MAPK, NOD-like receptor, and focal adhesion. Survival analyses of the 130 genes in the TCGA PCa database revealed that 10 of the deslanoside-downregulated genes (ITG2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, TMEM121, SOX18, CMPK2, and MAMDC4) inversely correlated, while one deslanoside-upregulated gene (RASD1) positively correlated, with disease-free survival in PCa patients. In addition, one deslanoside-downregulated gene (ENG) inversely correlated, while three upregulated genes (JUN, MXD1, and AQP3) positively correlated with overall survival in PCa patients. Some of the 15 genes have not been implicated in cancer before. These findings provide another candidate for repurposing cardiac glycosides for anticancer drugs. They also suggest that a diverse range of molecular events underlie deslanoside’s anticancer activity in PCa cells.

Progesterone (Pg)/progesterone receptor (PR) signaling drives mammary gland side-branching and alveologenesis, but the mechanisms through which Pg/PR signaling functions remain to be clarified. Using in vitro and in vivo models and histological and molecular analyses, we determined the role of Zfhx3 transcription factor in mammary gland development driven by Pg/PR signaling. Postnatal deletion of Zfhx3 in mouse mammary epithelial cells attenuated side-branching morphogenesis and alveologenesis. These effects were undetectable in the absence of Pg/PR signaling. During the estrus cycle, Zfhx3 expression corresponded to that of Pg, being at the highest level at the diestrus stage; Zfhx3 deletion inhibited mammary gland branching more potently at diestrus than estrus stage. Loss of Zfhx3 not only attenuated the expansion of stem/progenitor cells driven by Pg/PR signaling, but also impaired the function of Pg/PR signaling in the transcriptional activation of multiple genes. In addition, Pg/PR signaling significantly expanded PR- and Zfhx3-positive epithelial cells, and induced the physical association of ZFHX3 with PR. These findings establish Zfhx3 as an integral transcription factor of Pg/PR signaling in driving side-branching and alveologenesis during mammary gland development.

Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-β induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling., The therapies for bone metastatic prostate cancer are limited and the underlying mechanisms are unclear. Here, the authors show that bone derived TGF-β induces acetylation of KLF5 (Ac-KLF5), and Ac-KLF5 promotes prostate cancer bone metastasis and resistance to docetaxel by upregulating CXCR4.

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

Simple Summary Molecular signatures predictive of recurrence-free survival (RFS) and castration resistance are critical for treatment decision-making in prostate cancer (PCa), but the robustness of current signatures is limited. This study aims to identify castration-resistant PCa (CRPC)-associated genes and develop robust RFS and CRPC signatures. Among 287 genes differentially expressed between localized CRPC and hormone-sensitive PCa (HSPC) samples, 6 genes constituted a signature (CRPC-derived prognosis signature, CRPCPS) that predicted RFS. Moreover, a 3-gene panel derived from the 6 CRPCPS genes was capable of distinguishing CRPC from HSPC. The CRPCPS predicted RFS in 5/9 cohorts in the multivariate analysis and maintained prognostic in patients stratified by tumor stage, Gleason score, and lymph node metastasis status. It also predicted overall survival and metastasis-free survival. Notably, the signature was validated in another six independent cohorts. These findings suggest that these two signatures could be robust tools for predicting RFS and CRPC in clinical practice. Abstract Molecular signatures predictive of recurrence-free survival (RFS) and castration resistance are critical for treatment decision-making in prostate cancer (PCa), but the robustness of current signatures is limited. Here, we applied the Robust Rank Aggregation (RRA) method to PCa transcriptome profiles and identified 287 genes differentially expressed between localized castration-resistant PCa (CRPC) and hormone-sensitive PCa (HSPC). Least absolute shrinkage and selection operator (LASSO) and stepwise Cox regression analyses of the 287 genes developed a 6-gene signature predictive of RFS in PCa. This signature included NPEPL1, VWF, LMO7, ALDH2, NUAK1, and TPT1, and was named CRPC-derived prognosis signature (CRPCPS). Interestingly, three of these 6 genes constituted another signature capable of distinguishing CRPC from HSPC. The CRPCPS predicted RFS in 5/9 cohorts in the multivariate analysis and remained valid in patients stratified by tumor stage, Gleason score, and lymph node status. The signature also predicted overall survival and metastasis-free survival. The signature’s robustness was demonstrated by the C-index (0.55–0.74) and the calibration plot in all nine cohorts and the 3-, 5-, and 8-year area under the receiver operating characteristic curve (0.67–0.77) in three cohorts. The nomogram analyses demonstrated CRPCPS’ clinical applicability. The CRPCPS thus appears useful for RFS prediction in PCa.

Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells&rsquo, proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice, and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells&rsquo, proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

Simple Summary Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. The ZFHX3 transcription factor regulates mammary epithelial cells’ proliferation and differentiation by interacting with estrogen and progesterone receptors. Both these receptors play crucial roles in breast cancer development, but whether ZFHX3 also impacts breast cancer is unknown. In this study, the authors aim to determine if ZFHX3 promotes breast cancer cells’ proliferation and tumor growth and explore the underlying cellular and molecular mechanisms. Higher ZFHX3 expression is associated with worse patient survival in breast cancer, ZFHX3 promotes the proliferation and tumor growth of breast cancer cells, and several breast cancer stem cell factors appear to be involved in the role of ZFHX3 in breast cancer growth. The findings suggest that ZFHX3 is a novel oncogenic molecule promoting breast cancer development. Such a molecule could provide novel opportunities for the treatment of breast cancer. Abstract Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells’ proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice; and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells’ proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

Purpose Invasive micropapillary carcinoma (IMPC) of the breast has a high propensity for lymphovascular invasion and axillary lymph node metastasis and displays an ‘inside-out’ growth pattern, but the molecular mechanism of metastasis and tumor cell polarity reversal in IMPC is unclear. Methods and Patients: Luciferase reporter assays and western blotting were performed to confirm that the MTDH 3’UTR bound to miR-30c. Growth curves, tumor sphere formation assays, Transwell migration and invasion assays, mouse xenograft model and immunofluorescence were evaluated to investigate the effects of miR-30c and MTDH. MiRNA in situ hybridization (ISH) and immunohistochemistry (IHC) were carried out on IMPC patient tissues for miR-30c and MTDH expression, respectively. Results We found that miR-30c could directly target the MTDH (metadherin) 3’UTR. MiR-30c overexpression inhibited breast cancer cell proliferation, invasion and metastasis by targeting MTDH in vitro and in vivo. Moreover, miR-30c regulated IMPC cell polarity reversal by targeting MTDH. By in situ hybridization and immunohistochemistry analyses, miR-30c and MTDH were significantly correlated with tumor size, lymph nodule status and tumor grade, the ‘inside-out’ growth pattern, overall survival (OS) and disease-free survival (DFS) in IMPC patients (p

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Kr&uuml, ppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.

Background PSMA expression in the prostate epithelium is controlled by a cis-element, PSMA enhancer (PSME). PSME contains multiple binding sites for Sox proteins, and in this study, we identified Sox7 protein as a negative regulator of PSMA expression through its interaction with PSME. Methods The statistical correlation between Sox7 and PSMA mRNA expression was evaluated using five prostate cancer studies from cBioportal. In vitro and in vivo interaction between Sox7 and PSME was evaluated by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase reporter assay. Synthetic oligonucleotides were generated to define the sites in PSME that interact with Sox7 protein. Sox7 mutants were generated to identify the region of this protein required to regulate PSMA expression. Sox7 was also stably expressed in LNCaP/C4-2 and 22Rv1 cells to validate the regulation of PSMA expression by Sox7 in vivo. Results Sox7 mRNA expression negatively correlated with PSMA/FOLH1 and PSMAL/FOLH1B mRNA expression in Broad/Cornell, TCGA and MSKCC studies, but not in two studies containing only metastatic prostate tumors. PC-3 cells mostly expressed the 48.5 KDa isoform 2 of Sox7, and the depletion of this isoform did not restore PSMA expression. Ectopic expression of canonical, wild-type Sox7 in C4-2 and 22Rv1 cells suppressed PSMA protein expression. ChIP assay revealed that canonical Sox7 protein preferentially interacts with PSME in vivo, and EMSA identified the SOX box sites #2 and #4 in PSME as required for its interaction. Sox7 was capable of directly binding to PSME and suppressed PSME-mediated transcription. The NLS regions of Sox7, but not its β-catenin interacting motif, are essential for this suppressing activity. Furthermore, restoration of wild-type Sox7 expression but not Sox7-NLS mutant in Sox7-null prostate cancer cell lines suppressed PSMA expression. Conclusions The inactivation of canonical Sox7 is responsible for the upregulated expression of PSMA in non-metastatic prostate cancer.

The novel zinc finger protein 121 (ZNF121) has been demonstrated to physically and functionally associate with the MYC oncoprotein to regulate cell proliferation and likely breast cancer development. To further understand how ZNF121 functions in cell proliferation and carcinogenesis, we identified and characterized the interaction of ZNF121 with zinc finger and BRCA1-interacting protein with a KRAB domain 1 (ZBRK1), a breast and ovarian cancer susceptibility protein 1 (BRCA1)-interacting protein, using the yeast two-hybrid assay and other approaches. We also found that ZNF121 bound to BRCA1. Functionally, ZFN121 suppressed the expression of ANG1 and HMGA2, two common downstream targets of ZBRK1 and BRCA1. Interestingly, ZNF121 also regulated the expression of BRCA1 and ZBRK1. These findings suggest that ZNF121 is likely a member of the BRCA1/CtIP/ZBRK1 repressor complex that plays a role in breast cancer.

Krüppel-like factor 5 (KLF5) both suppresses and promotes tumor growth depending on cellular context. The mechanisms underlying tumor promotion could be targetable for therapy. Although a number of transcriptional targets of KLF5 have been identified and implicated in KLF5-mediated tumor growth, how KLF5 regulates these genes remains to be addressed. Here we performed coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the TSU-Pr1 bladder cancer cell line, in which KLF5 is shown to promote tumor growth, to identify KLF5-interacting nuclear proteins that are necessary for KLF5's tumor promoting function. LC-MS/MS revealed 122 potential KLF5 binding proteins in the nuclear proteins precipitated by the KLF5 antibody, and the top nine candidates included AHNAK, TFAM, HSDL2, HNRNPC, CINP, IST1, FBL, PABPC1 and SNRNP40. SRB assays of these nine proteins indicated that silencing CINP had the most potent inhibitory effect on cell growth in KLF5-expressing cells but did not affect parental TSU-Pr1 cells. Further analyses not only confirmed the physical interaction between KLF5 and CINP, also demonstrated that knockdown of CINP attenuated the effects of KLF5 on cell cycle progression, apoptosis and tumorigenesis. Silencing CINP also attenuated the effect of KLF5 on the expression of a number of genes and signaling pathways, including cell cycle regulator Cyclin D1 and apoptosis-related Caspase 7. These results suggest that CINP is a cofactor of KLF5 that is crucial for the promotion of tumor growth, and that the KLF5-CINP interaction could be a novel therapeutic target for inhibiting KLF5-promoted tumor growth.

Krüppel-like factor 5 (KLF5) is a basic transcription factor that regulates diverse cellular processes during tumor development. Acetylation of KLF5 at lysine 369 (K369) reverses its function from promoting to suppressing cell proliferation and tumor growth. In this study, we examined the regulation of KLF5 by histone deacetylases in the prostate cancer cell line DU 145. While confirming the functions of HDAC1/2 in KLF5 deacetylation and the promotion of cell proliferation, we found that the knockdown of HDAC1/2 upregulated KLF5 protein but not KLF5 mRNA, and the increase in KLF5 protein level by silencing HDAC1/2 was at least in part due to decreased proteasomal degradation. Deacetylase activity was required for HDAC1/2-mediated KLF5 degradation, and mutation of KLF5 to an acetylation-mimicking form prevented its degradation, even though the mutation did not affect the binding of KLF5 with HDAC1/2. Mutation of K369 to arginine, which prevents acetylation, did not affect the binding of KLF5 to HDAC1 or the response of KLF5 to HDAC1/2-promoted degradation. These findings provide a novel mechanistic association between the acetylation status of KLF5 and its protein stability. They also suggest that maintaining KLF5 in a deacetylated form may be an important mechanism by which KLF5 and HDACs promote cell proliferation and tumor growth.

Summary Lifestyle factors, including diet, play an important role in the survival of cancer patients. However, the molecular mechanisms underlying pathogenic links between diet and particular oncogenic mutations in human cancers remain unclear. We recently reported that the ketone body acetoacetate selectively enhances BRAF V600E mutant-dependent MEK1 activation in human cancers. Here we show that a high-fat ketogenic diet increased serum levels of acetoacetate, leading to enhanced tumor growth potential of BRAF V600E-expressing human melanoma cells in xenograft mice. Treatment with hypolipidemic agents to lower circulating acetoacetate levels or an inhibitory homolog of acetoacetate, dehydroacetic acid, to antagonize acetoacetate-BRAF V600E binding attenuated BRAF V600E tumor growth. These findings reveal a signaling basis underlying a pathogenic role of dietary fat in BRAF V600E-expressing melanoma, providing insights into the design of conceptualized "precision diets" that may prevent or delay tumor progression based on an individual's specific oncogenic mutation profile.

MYC is a potent oncoprotein that modulates multiple cellular processes including proliferation, apoptosis, differentiation, stemness, senescence, and migration. Functioning primarily as a transcriptional factor, MYC interacts with a large number of proteins, and identification and characterization of MYC-interacting proteins are important for understanding how MYC functions. In this study, we used different systems to demonstrate that a novel zinc finger transcription factor, ZNF121, physically interacted with MYC, and the interaction involved their N-terminal regions. Overexpression of ZNF121 increased, while its knockdown decreased, the expression of MYC in multiple epithelial cell lines, and MYC had similar effects on ZNF121 expression. An expression correlation was also detectable in a panel of epithelial cell lines and a cohort of human breast cancer tissues. Functionally, knockdown of ZNF121 in several breast epithelial cell lines attenuated the expressions of MYC and its target genes (e.g., EGR1, CDC2, and nucleolin) and slowed cell proliferation, accompanied by cell cycle arrest in the G1 phase and expression alteration of cell cycle regulators (cyclin D1, p14 and p21). Analysis of publically available databases showed that ZNF121 expression is up-regulated in human breast cancer, and the up-regulation significantly associates with worse patient survival in the luminal A subtype of breast cancer. These findings establish ZNF121 as a MYC-interacting protein with functional effects on MYC and cell proliferation.

Zinc finger homeobox 3 (ZFHX3) is a transcription factor that regulates multiple cellular processes including cell proliferation, differentiation and neoplastic development. It is also involved in the function of steroid hormones estrogen and progesterone and the peptide hormone prolactin in mammary epithelial cells. In this study, we investigated whether and how ZFHX3 regulates intracellular calcium homeostasis in mammary epithelial cells. We found that ZFHX3 affected both store operated calcium entry and store independent calcium entry (SOCE and SICE). Simultaneously, the expression of the calcium channel TRPV6 was regulated by ZFHX3, as demonstrated by expression analysis and luciferase reporter assay. In cells with knockdown of ZFHX3, calcium entry was partially rescued by the overexpression of wild type but not the pore mutants of TRPV6. In addition, overexpression of TRPV6 promoted differentiation of the MCF10A mammary epithelial cells in three-dimensional culture, which is consistent with our previous findings that ZFHX3 is essential for mammary gland differentiation. These findings suggest that ZFHX3 plays an important role in intracellular calcium homeostasis in mammary epithelial cells, at least in part, by regulating TRPV6.

Both estrogen receptor 2 (ESR2, also known as estrogen receptor beta (ERβ)) and the zinc-finger homeobox 3 (ZFHX3, also known as ATBF1 for AT motif-binding factor 1) modulate prostate development and suppress prostatic tumorigenesis in mice. ZFHX3 is integral to proper functions of ESR1 (i.e., estrogen receptor alpha (ERα)), which belongs to the same family of proteins as ESR2, but is hardly expressed in prostate epithelial cells. It is not clear how ZFHX3 suppresses prostatic tumorigenesis. In this study, we investigated whether ZFHX3 and ERβ functionally interact with each other in the suppression of prostatic tumorigenesis. In two androgen receptor (AR)-positive prostate cancer cell lines, C4-2B and LNCaP, we first validated ERβ’s tumor suppressor activity indicated by the inhibition of cell proliferation and repression of MYC expression. We found that loss of ZFHX3 increased cell proliferation and MYC expression, and downregulation of MYC was necessary for ZFHX3 to inhibit cell proliferation in the same cell lines. Importantly, loss of ZFHX3 prevented ERβ from suppressing cell proliferation and repressing MYC transcription. Biochemically, ERβ and ZFHX3 physically interacted with each other and they both occupied the same region of the common MYC promoter, even though ZFHX3 also bound to another region of the MYC promoter. Higher levels of ZFHX3 and ERβ in human prostate cancer tissue samples correlated with better patient survival. These findings establish MYC repression as a mechanism for ZFHX3’s tumor suppressor activity and ZFHX3 as an indispensable factor for ERβ’s tumor suppressor activity in prostate cancer cells. Our data also suggest that intact ZFHX3 function is required for using ERβ-selective agonists to effectively treat prostate cancer.

Although emerging evidence suggests positive association of arsenic (As) or sulfur dioxide (SO2) exposure with human diseases, reports concerning the effects of co-exposure of As and SO2 are lacking. Moreover, there is insufficient information in the literature about As and SO2 co-exposure to renal injury. In this study, we focus on the environmental problems of excessive As and SO2 that co-exist in many coal consumption areas. We used both C57BL/6 mice and 293T cells to detect toxicities of As and SO2 exposure alone or in combination. Our results showed that co-exposure significantly increased the hazard compared with exposure to As or SO2 alone. Mouse kidney tissue slices showed that co-exposure caused more severe diffuse sclerosing glomerulonephritis than As and SO2 exposure alone. Meanwhile experiments showed that apoptosis was aggravated by co-exposure of As and SO2 in 293T cells. Because As and SO2 cause cell toxicity through increasing oxidative stress, next we detected ROS and other oxidative stress parameters, and the results showed oxidative stress was increased by co-exposure compared with the other three groups. The expression levels of downstream genes in the NF-κB and caspase pathways were higher in the co-exposure group than in the groups of As or SO2 exposure alone in mice and 293T cells. Based on the above results, co-exposure could induce higher toxicity in vitro and in vivo compared with single exposure to As or SO2, indicating that people living in places that contaminated by As and SO2 may have higher chance to get renal injury.

The zinc finger homeobox 3 (ZFHX3, also named ATBF1 for AT motif binding factor 1) is a transcription factor that suppresses prostatic carcinogenesis and induces neuronal differentiation. It also interacts with estrogen receptor α to inhibit cell proliferation and regulate pubertal mammary gland development in mice. In the present study, we examined whether and how Zfhx3 regulates lactogenic differentiation in mouse mammary glands. At different stages of mammary gland development, Zfhx3 protein was expressed at varying levels, with the highest level at lactation. In the HC11 mouse mammary epithelial cell line, an in vitro model of lactogenesis, knockdown of Zfhx3 attenuated prolactin-induced β-casein expression and morphological changes, indicators of lactogenic differentiation. In mouse mammary tissue, knock-out of Zfhx3 interrupted lactogenesis, resulting in underdeveloped glands with much smaller and fewer alveoli, reduced β-casein expression, accumulation of large cytoplasmic lipid droplets in luminal cells after parturition, and failure in lactation. Mechanistically, Zfhx3 maintained the expression of Prlr (prolactin receptor) and Prlr-Jak2-Stat5 signaling activity, whereas knockdown and knock-out of Zfhx3 in HC11 cells and mammary tissues, respectively, decreased Prlr expression, Stat5 phosphorylation, and the expression of Prlr-Jak2-Stat5 target genes. These findings indicate that Zfhx3 plays an essential role in proper lactogenic development in mammary glands, at least in part by maintaining Prlr expression and Prlr-Jak2-Stat5 signaling activity.

Luminal A breast cancer usually responds to hormonal therapies but does not benefit from chemotherapies, including microtubule-targeted paclitaxel. MicroRNAs could play a role in mediating this differential response. In this study, we examined the role of micro RNA 100 (miR-100) in the sensitivity of breast cancer to paclitaxel treatment. We found that while miR-100 was downregulated in both human breast cancer primary tumors and cell lines, the degree of downregulation was greater in the luminal A subtype than in other subtypes. The IC50 of paclitaxel was much higher in luminal A than in basal-like breast cancer cell lines. Ectopic miR-100 expression in the MCF-7 luminal A cell line enhanced the effect of paclitaxel on cell cycle arrest, multinucleation, and apoptosis, while knockdown of miR-100 in the MDA-MB-231 basal-like line compromised these effects. Similarly, overexpression of miR-100 enhanced the effects of paclitaxel on tumorigenesis in MCF-7 cells. Rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR), a target of miR-100, also sensitized MCF-7 cells to paclitaxel. Gene set enrichment analysis showed that genes that are part of the known paclitaxel-sensitive signature had a significant expression correlation with miR-100 in breast cancer samples. In addition, patients with lower levels of miR-100 expression had worse overall survival. These results suggest that miR-100 plays a causal role in determining the sensitivity of breast cancers to paclitaxel treatment.

Purpose: In this study, we explored the role of KLF5 in TGF-β-induced drug resistance in prostate cancer cells to identify novel therapeutic targets. Procedures: We used DU 145 and PC-3 prostate cancer cell lines in in vitro cell survival assay, colony survival assay, and xenograft mouse model to measure docetaxel (DTX) resistance. TGF-β1, the SB505124 TGF-β receptor I inhibitor, and the ABT-199 Bcl-2 inhibitor were used to determine if Bcl-2 mediates TGFβ induced docetaxel resistance. To measure the function of KLF5 and its acetylation at lysine 369 in drug resistance, stable cell lines were constructed with different forms of KLF5 after the endogenous KLF5 was deleted with the CRISPR-Cas9 system. Results: We found that TGF-β induced DTX resistance depends on KLF5 expression. As a downstream molecule of TGF-β signaling, acetylated KLF5 at lysine 369 mediated TGF-β-induced DTX resistance. Moreover, acetylated KLF5 induced DTX resistance in xenograft mouse model. Mechanistically, we found that TGF-β and acetylated KLF5 upregulated Bcl-2 transcription, and inhibition of Bcl-2 sensitized cells to DTX. In addition, DTX promoted Bcl-2 degradation via a proteasome dependent pathway, and TGF-β signaling stabilized Bcl-2 via inhibition of Bcl-2 ubiquitination. Lastly, the TGF-β/acetylated KLF5/Bcl-2 signaling axis induced survival disadvantage in prostate cancer patients. Conclusions: TGF-β induces transcriptional upregulation of Bcl-2 through acetylated KLF5 at lysine 369, and TGF-β stabilizes Bcl-2 via inhibition of DTX-induced Bcl-2 ubiquitination. The TGF-β-acetylated KLF5-Bcl-2 signaling axis is thus a novel mechanism mediating DTX resistance in prostate cancer. Citation Format: Yixiang Li, Baotong Zhang, Jin-Tang Dong. TGF-β-acetylated KLF5-Bcl-2 signaling mediates docetaxel resistance in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5287.

Advanced prostate cancer most commonly spreads to bones. Although denosumab or zoledronic acid is recommended for prostate cancer patients with bone metastases to prevent or delay skeletal-related events, no significant benefits in terms of cancer-specific and overall survival have been observed. Some chemotherapies are available for the treatment of bone metastasis, but eventually all such tumors develop resistance. Additionally, the underlying mechanisms of prostate cancer bone metastasis and its resistance to chemotherapy are still poorly understood. In this study, we report that TGF-β-induced acetylation of the basic transcription factor Krüppel-like factor 5 (KLF5) frequently occurred in prostate cancer cells located in both the mouse and human bone microenvironments. Using multiple in vitro and in vivo systems, we demonstrated that tumor cells expressing acetylated KLF5 (Ac-KLF5) enhanced prostate cancer-induced bone lesions by promoting osteoclast differentiation. As revealed by RNA-Seq and ChIP-Seq analyses, Ac-KLF5 transcriptionally activated CXCR4 expression, the silence of which attenuated Ac-KLF5-enhanced osteoclast differentiation. A further screening on the paracrine signaling indicates that IL-11 mediated the promoting effect of the Ac-KLF5/CXCR4 axis on osteoclast differentiation. These findings indicate that the TGF-β/Ac-KLF5/CXCR4/IL-11 signaling axis from bone to tumor and then to bone again is a crucial pathway responsible for TGF-β mediated vicious cycle in bone metastasis. More importantly, targeting CXCR4 using FDA approved drugs AMD3100 selectively sensitizes Ac-KLF5-expressing tumors and bone metastases to docetaxel treatment. These findings provide a rationale for targeting Ac-KLF5/CXCR4 signaling as a novel therapeutic strategy in advanced prostate cancer patients with bone metastases. Citation Format: Baotong Zhang, Yixiang Li, Qiao Wu, Lin Xie, Benjamin Barwick, Jin-Tang Dong. KLF5 acetylation promotes bone metastatic growth of prostate cancer by activating CXCR4/IL-11 and subsequent osteoclast differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3840.

Acetylated Kruppel-like factor 5 (KLF5) is essential for transforming growth factor-β (TGF-β) to properly regulate gene transcription in the inhibition of cell proliferation and tumor growth. Ras oncogenic signaling can convert TGF-β from a tumor suppressor to a tumor promoter; however, its ability to utilize the KLF5 transcription factor to modulate TGF-β functions is still unknown. Therefore, in this study, we sought to determine whether Ras signaling altered TGF-β-induced KLF5 acetylation and the assembly of the p300-KLF5-SMADs transcriptional complex in gene regulation. Not only did we determine that Ras signaling inhibited TGF-β-induced KLF5 acetylation and interfered with TGF-β function in p15 induction and Myc repression, but also TGF-β-induced SMAD3 C-terminal region phosphorylation was necessary for TGF-β to induce KLF5 acetylation. Moreover, Ras activation further interrupted the interactions amongst p300, KLF5, and SMAD4, as well as the binding of p300-KLF5-SMADs complex onto the TGF-β-responsive promoter elements for both p15 and Myc. These findings suggested that KLF5 mediated the crosstalk between TGF-β and Ras signaling, and that suppression of TGF-β-induced KLF5 acetylation by Ras activation; this altered TGF-β-induced assembly of p300-KLF5-SMADs complex onto gene promoters to convert the function of TGF-β in gene regulation.

The elucidation of molecular events that confer tamoxifen resistance to estrogen receptor α (ER) positive breast cancer is of major scientific and therapeutic importance. Here, we report that LEM4 overexpression renders ER+ breast cancer cells resistant to tamoxifen by activating the cyclin D-CDK4/6 axis and the ERα signaling. We show that LEM4 overexpression accelerates tumor growth. Interaction with LEM4 stabilizes CDK4 and Rb, promotes Rb phosphorylation and the G1/S phase transition. LEM4 depletion or combined tamoxifen and PD0332991 treatment significantly reverses tamoxifen resistance. Furthermore, LEM4 interacts with and stabilizes both Aurora-A and ERα, promotes Aurora-A mediated phosphorylation of ERα-Ser167, leading to increase in ERα DNA-binding and transactivation activity. Elevated levels of LEM4 correlates with poorer relapse-free survival in patients with ER+ breast cancer undergoing endocrine therapy. Thus, LEM4 represents a prognostic marker and an attractive target for breast cancer therapeutics. Functional antagonism of LEM4 could overcome tamoxifen resistance., Tamoxifen resistance is a major problem in the treatment of ERa positive breast cancer. Here, the authors show that LEM4 confers tamoxifen resistance by activating the cyclin D-CDK4/6-Rb axis and ERa signaling in endocrine-resistant breast cancer.

Abnormal expression of TTK kinase has been associated with the initiation, progression, and therapeutic resistance of breast and other cancers, but its roles remain to be clarified. In this study, we examined the role of TTK in triple negative breast cancer (TNBC), and found that higher TTK expression correlated with mesenchymal and proliferative phenotypes in TNBC cells. Pharmacologic inhibition and genomic silencing of TTK not only reversed the epithelial-to-mesenchymal transition (EMT) in TNBC cells, but also increased the expression of KLF5, an effector of TGF-β signaling and inhibitor of EMT. In addition, TTK inhibition decreased the expression of EMT-associated micro-RNA miR-21 but increased the expression of miR-200 family members and suppressed TGF-β signaling. To test if upregulation of KLF5 plays a role in TTK-induced EMT, TTK and KLF5 were silenced simultaneously, which reversed the decreased EMT caused by loss of TTK. Consistently, the decrease in miR-21 expression and increase in miR-200 expression caused by TTK silencing were rescued by loss of KLF5. Altogether, this study highlights a novel role and signaling pathway for TTK in regulating EMT of TN breast cancer cells through TGF-β and KLF5 signaling, highlighting targetable signaling pathways for TTK inhibitors in aggressive breast cancer.

Dysregulation of microRNA expression plays a pivotal role in the initiation and progression of a variety of human carcinomas including prostate cancer. Our previous studies have demonstrated that the silence of miR-203 contributes to the invasiveness of malignant breast cancer cells by targeting SNAI2. However, the effects and underlying mechanisms of miR-203/SNAI2 axis in prostate cancer have not been elucidated. The aim of this study is to explore the effects of miR-203/SNAI2 axis on the biological characteristics of prostate carcinomas both in vitro and in vivo. We found that miR-203 was significantly downregulated in prostate cancer cell lines compared with immortalized prostate epithelial cells using semi-quantitative PCR and real-time PCR, as well as in clinical prostate cancer tissues compared to normal tissues using TCGA analysis. Functionally, miR-203 inhibited prostate cancer cell proliferation, migration, endothelial cell tube formation and cancer stemness in vitro. Meanwhile, overexpression of miR-203 suppressed SNAI2 expression both in DU145 and PC3 cells. In addition, the in vivo study showed that miR-203 suppressed tumorigenicity, metastasis and angiogenesis of DU145 cells. Ectopic expression of SNAI2 rescued the inhibitory effects of miR-203 both in vitro and in vivo. Importantly, the EMT markers CDH1 and VIMENTIN were modulated by the miR-203/SNAI2 axis. Furthermore, the GSK-3β/β-CATENIN signal pathway was suppressed by miR-203 and could be reactivated by SNAI2. Taken together, this research unveiled the function of miR-203/SNAI2 axis in tumorigenesis, angiogenesis, stemness, metastasis and GSK-3β/β-CATENIN signal pathway in prostate cancer and gave insights into miR-203/SNAI2-targeting therapy for prostate cancer patients. © 2018 IUBMB Life, 70(3):224-236, 2018.

The phosphatase and tensin homolog (PTEN) and the zinc finger homeobox 3 (ZFHX3)/AT-motif binding factor 1 (ATBF1) genes have been established as tumor suppressor genes in prostate cancer by their frequent deletions and mutations in human prostate cancer and by the formation of mouse prostatic intraepithelial neoplasia (mPIN) or tumor by their deletions in mouse prostates. However, whether ZFHX3/ATBF1 deletion together with PTEN deletion facilitates prostatic tumorigenesis is unknown. In this study, we simultaneously deleted both genes in mouse prostatic epithelia and performed histological and molecular analyses. While deletion of one Pten allele alone caused low-grade (LG) mPIN as previously reported, concurrent deletion of Zfhx3/Atbf1 promoted the progression to high-grade (HG) mPIN or early carcinoma. Zfhx3/Atbf1 and Pten deletions together increased cell proliferation, disrupted the smooth muscle layer between epithelium and stroma, and increased the number of apoptotic cells. Deletion of both genes also accelerated the activation of Akt and Erk1/2 oncoproteins. These results suggest an additive effect of ZFHX3/ATBF1 and PTEN deletions on the development and progression of prostate neoplasia.