A role of SIPL1/SHARPIN in promoting resistance to hormone therapy in breast cancer.

SIPL1 inhibits PTEN function and stimulates NF-κB signaling; both processes contribute to resistance to hormone therapy in estrogen receptor positive breast cancer (ER+ BC). However, whether SIPL1 promotes tamoxifen resistance in BC remains unclear. We report here that SIPL1 enhances tamoxifen resistance in ER+ BC. Overexpression of SIPL1 in MCF7 and TD47 cells conferred tamoxifen resistance. In MCF7 cell-derived tamoxifen resistant (TAM-R) cells, SIPL1 expression was upregulated and knockdown of SIPL1 in TAM-R cells re-sensitized the cells to tamoxifen. Furthermore, xenograft tumors produced by MCF7 SIPL1 cells but not by MCF7 empty vector cells resisted tamoxifen treatment. Collectively, we demonstrated a role of SIPL1 in promoting tamoxifen resistance in BC. Increases in AKT activation and NF-κB signaling were detected in both MCF7 SIPL1 and TAM-R cells; using specific inhibitors and unique SIPL1 mutants to inhibit either pathway significantly reduced tamoxifen resistance. A SIPL1 mutant defective in activating both pathways was incapable of conferring resistance to tamoxifen, showing that both pathways contributed to SIPL1-derived resistance to tamoxifen in ER+ BCs. Using the Curtis dataset of breast cancer (n=1980) within the cBioPortal database, we examined a correlation of SIPL1 expression with ER+ BC and resistance to hormone therapy. SIPL1 upregulation strongly associates with reductions in overall survival in BC patients, particularly in patients with hormone naïve ER+ BCs. Taken together, we provide data suggesting that SIPL1 contributes to promote resistance to tamoxifen in BC cells through both AKT and NF-κB actions.

CYB5D2 displays tumor suppression activities towards cervical cancer.

Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cell's ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.

Changes in PKM2 associate with prostate cancer progression.

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis, the dominant metabolic pathway utilized by cancer cells. To determine the association of PKM2 with prostate cancer (PC), we examined 29 primary PC and three lymph node metastatic tumors; elevation of PKM2 was observed in Gleason 8-10 tumors compared to Gleason 6-7 carcinomas. High PKM2 was detected by immunohistochemistry in more aggressive xenograft tumors derived from PC stem-like cells (PCSCs) compared to those produced from non-PCSCs. While PCSCs and non-PCSCs expressed comparable levels of PKM2, distinct posttranslational modifications were observed. Collectively, upregulation and specific modification to PKM2 associate with PC progression.

Downregulation of CYB5D2 is associated with breast cancer progression.

We report here that CYB5D2 is associated with tumor suppression function in breast cancer (BC). CYB5D2 expression was significantly reduced in tamoxifen resistant MCF7 cells and in MCF7 cell-derived xenografts treated with TAM. CYB5D2 overexpression induced apoptosis in MCF7 cells; CYB5D2 knockdown enhanced MCF7 cell proliferation. Using the TCGA and Curtis datasets within the Oncomine database, CYB5D2 mRNA expression was downregulated in primary BCs vs breast tissues and HER2-positive or triple negative BCs vs estrogen receptor (ER)-positive BCs. Using the TCGA and Metabric datasets (n = 817 and n = 2509) within cBioPortal, 660 and 4891 differentially expressed genes (DEGs) in relation to CYB5D2 were identified. These DEGs were enriched in pathways governing cell cycle progression, progesterone-derived oocyte maturation, oocyte-meiosis, estrogen-mediated S-phase entry, and DNA metabolism. CYB5D2 downregulation decreased overall survival (OS, p = 0.0408). A CYB5D2-derived 21-gene signature was constructed and robustly correlated with OS shortening (p = 5.72e-12), and independently predicted BC deaths (HR = 1.28; 95% CI 1.08-1.52; p = 0.004) once adjusting for known clinical factors. CYB5D2 reductions displayed relationship with mutations in PIK3CA, GATA3, MAP3K1, CDH1, TP53 and RB1. Impressively, 85% (560/659) of TP53 mutations occurred in the 21-gene signature-positive BC. Collectively, we provide the first evidence that CYB5D2 is a candidate tumor suppressor of BC.

Polycomb complex protein BMI1 confers resistance to tamoxifen in estrogen receptor positive breast cancer.

We report that BMI1 promotes tamoxifen resistance in estrogen receptor (ER)-positive breast cancer (BC). BMI1 overexpression conferred MCF7 and TD47 cells resistance to tamoxifen; BMI1 knockdown sensitized the process. In MCF7-derived tamoxifen resistant cells, BMI1 expression was upregulated and BMI1 knockdown reduced the resistance. BMI1 is an oncogene; its oncogenic activity is attributed to BMI1-stimulated E3 ubiquitin ligase activity, a process that requires BMI1's ring finger (RF) domain. However, a BMI1 mutant without RF conferred tamoxifen resistance. Tamoxifen significantly reduced the growth of xenografts derived from MCF7 cells, but accelerated the growth of tumors produced by BMI1 overexpressing MCF7 cells. BMI1 enhances the pathways that promote resistance to endocrine therapy, including ER, androgen receptor, and MUC1. In patients with ER + BCs (n = 177), BMI1 expression was associated with BC recurrence. In the Curtis dataset consisting of ER + BCs (n = 1506) and ER- BCs (n = 474; cBioPortal), upregulations in BMI1 mRNA expression were correlated with ER + BCs; the upregulation was associated with a set of differentially expressed genes (DEGs). These DEGs were enriched with reductions in immunological processes, indicating a role of BMI1 in downregulation of the immune surveillance.

Signatures derived from increase in SHARPIN gene copy number are associated with poor prognosis in patients with breast cancer.

We report three signatures produced from SHARPIN gene copy number increase (GCN-Increase) and their effects on patients with breast cancer (BC). In the Metabric dataset (n = 2059, cBioPortal), SHARPIN GCN-Increase occurs preferentially or mutual exclusively with mutations in TP53, PIK3CA, and CDH1. These genomic alterations constitute a signature (SigMut) that significantly correlates with reductions in overall survival (OS) in BC patients (n = 1980; p = 1.081e - 6). Additionally, SHARPIN GCN-Increase is associated with 4220 differentially expressed genes (DEGs). These DEGs are enriched in activation of the pathways regulating cell cycle progression, RNA transport, ribosome biosynthesis, DNA replication, and in downregulation of the pathways related to extracellular matrix. These DEGs are thus likely to facilitate the proliferation and metastasis of BC cells. Additionally, through forward (FWD) and backward (BWD) stepwise variate selections among the top 160 downregulated and top 200 upregulated DEGs using the Cox regression model, a 6-gene (SigFWD) and a 50-gene (SigBWD) signature were derived. Both signatures robustly associate with decreases in OS in BC patients within the Curtis (n = 1980; p = 6.16e - 11 for SigFWD; p = 1.06e - 10, for SigBWD) and TCGA cohort (n = 817; p = 4.53e - 4 for SigFWD and p = 0.00525 for SigBWD). After adjusting for known clinical factors, SigMut (HR 1.21, p = 0.0297), SigBWD (HR 1.25, p = 0.0263), and likely SigFWD (HR 1.17, p = 0.062) remain independent risk factors of BC deaths. Furthermore, the proportion of patients positive for these signatures is significantly increased in ER -, Her2-enriched, basal-like, and claudin-low BCs compared to ER + and luminal BCs. Collectively, these SHARPIN GCN-Increase-derived signatures may have clinical applications in management of patients with BC.

Overexpression of MUC1 and Genomic Alterations in Its Network Associate with Prostate Cancer Progression.

We investigate the association of MUC1 with castration-resistant prostate cancer (CRPC), bone metastasis, and PC recurrence. MUC1 expression was studied in patient-derived bone metastasis and CRPCs produced by prostate-specific PTEN-/- mice and LNCaP xenografts. Elevations in MUC1 expression occur in CRPC. Among nine patients with hormone-naïve bone metastasis, eight express MUC1 in 61% to 100% of PC cells. Utilizing cBioPortal PC genomic data, we organized a training (n=300), testing (n=185), and validation (n=194) cohort. Using the Cox model, a nine-gene signature was derived, including eight genes from a MUC1-related network (APC, CTNNB1/ß-catenin, GALNT10, GRB2, LYN, SIGLEC1, SOS1, and ZAP70) and FAM84B. Genomic alterations in these genes reduce disease-free survival (DFS) in the training (P=.00161), testing (P=.00699), entire (training+testing, P=5.557e-5), and a validation cohort (P=3.326e-5). The signature independently predicts PC recurrence [hazard ratio (HR)=1.731; 95% confidence interval (CI): 1.104-2.712; P=.0167] after adjusting for known clinical factors and stratifies patients with high risk of PC recurrence using the median (HR 2.072; 95% CI: 1.245-3.450, P=.0051) and quartile 3 (HR 3.707, 95% CI: 1.949-7.052, P=6.51e-5) scores. Several novel ß-catenin mutants are identified in PCs leading to a rapid onset of death and recurrence. Genomic alterations in APC and CTNNB1/ß-catenin reduce DFS in two independent PC cohorts (n=485, P=.0369; n=84, P=.0437). The nine-gene signature also associates with reductions in overall survival (P=.0458) and DFS (P=.0163) in melanoma patients (n=367). MUC1 upregulation is associated with CRPC and bone metastasis. A nine-gene signature derived from a MUC1 network predicts PC recurrence.

Upregulation of FAM84B during prostate cancer progression.

Although the FAM84B gene lies within chromosome 8q24, a locus frequently altered in prostate cancer (PC), its alteration during prostate tumorigenesis has not been well studied. We report here FAM84B upregulation in DU145 cell-derived prostate cancer stem-like cells (PCSLCs) and DU145 cell-produced lung metastases compared to subcutaneous xenograft tumors. FAM84B protein was detected in bone metastases and primary PCs. Nanostring examination of 7 pairs of tumor adjacent normal and PC tissues revealed elevations in FAM84B mRNA levels in all carcinomas. Furthermore, through analysis of FAM84B expression using large datasets within the Gene Expression Omnibus and OncomineTM database, we demonstrate significant increases in FAM84B mRNA in 343 primary PCs versus 181 normal tissues, and elevations in the FAM84B gene copy number (GCN) in 171 primary PCs versus 61 normal tissues. While FAM84B was not detected at higher levels via immunohistochemistry in high grade (Gleason score/GS 8-10) tumors compared to GS6-7 PCs, analyses of FAM84B mRNA and GCN using datasets within the cBioPortal database demonstrated FAM84B upregulation in 12% (67/549) of primary PCs and 18% (73/412) of metastatic castration resistant PCs (mCRPCs), and GCN increases in 4.8% (26/546) of primary PCs and 26% (121/467) of mCRPCs, revealing an association of the aforementioned changes with CRPC development. Of note, an increase in FAM84B expression was observed in xenograft CRPCs produced by LNCaP cells. Furthermore, FAM84B upregulation and GCN increases correlate with decreases in disease free survival and overall survival. Collectively, we demonstrate a novel association of FAM84B with PC tumorigenesis and CRPC progression.

Dataset on the effects of CYB5D2 on the distribution of HeLa cervical cancer cell cycle.

We have recently reported that CYB5D2 plays a role in suppression of cervical cancer tumorigenesis, "CYB5D2 displays tumor suppression activities towards cervical cancer" [1]. We provide the accompany data here describing the effects of CYB5D2 overexpression and addition of recombinant CYB5D2 on HeLa cell cycle distribution. Furthermore, we will present the conditions used to specifically determine CYB5D2 expression in primary cervical and cervical cancer tissues using immunohistochemistry (IHC) and the patient cohort involved in assessing the CYB5D2 protein levels in primary cervical and cervical cancer tissues.

Neural Cell Adhesion Protein CNTN1 Promotes the Metastatic Progression of Prostate Cancer.

Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies.

PKM2 contributes to cancer metabolism.

Reprogramming of cell metabolism is essential for tumorigenesis, and is regulated by a complex network, in which PKM2 plays a critical role. PKM2 exists as an inactive monomer, less active dimer and active tetramer. While dimeric PKM2 diverts glucose metabolism towards anabolism through aerobic glycolysis, tetrameric PKM2 promotes the flux of glucose-derived carbons for ATP production via oxidative phosphorylation. Equilibrium of the PKM2 dimers and tetramers is critical for tumorigenesis, and is controlled by multiple factors. The PKM2 dimer also promotes aerobic glycolysis by modulating transcriptional regulation. We will discuss the current understanding of PKM2 in regulating cancer metabolism.

Prostate Cancer Stem-like Cells Contribute to the Development of Castration-Resistant Prostate Cancer.

Androgen deprivation therapy (ADT) has been the standard care for patients with advanced prostate cancer (PC) since the 1940s. Although ADT shows clear benefits for many patients, castration-resistant prostate cancer (CRPC) inevitably occurs. In fact, with the two recent FDA-approved second-generation anti-androgens abiraterone and enzalutamide, resistance develops rapidly in patients with CRPC, despite their initial effectiveness. The lack of effective therapeutic solutions towards CRPC largely reflects our limited understanding of the underlying mechanisms responsible for CRPC development. While persistent androgen receptor (AR) signaling under castration levels of serum testosterone (<50 ng/mL) contributes to resistance to ADT, it is also clear that CRPC evolves via complex mechanisms. Nevertheless, the physiological impact of individual mechanisms and whether these mechanisms function in a cohesive manner in promoting CRPC are elusive. In spite of these uncertainties, emerging evidence supports a critical role of prostate cancer stem-like cells (PCSLCs) in stimulating CRPC evolution and resistance to abiraterone and enzalutamide. In this review, we will discuss the recent evidence supporting the involvement of PCSLC in CRPC acquisition as well as the pathways and factors contributing to PCSLC expansion in response to ADT.

A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response.

BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

Factors Promoting Tamoxifen Resistance in Breast Cancer via Stimulating Breast Cancer Stem Cell Expansion.

Estrogen receptor-alpha positive (ER(+)) breast cancer constitutes 70-75% of the disease incidence. Tamoxifen has been the basis of endocrine therapy for patients with ER(+) breast cancer for more than three decades. The treatment reduces the annual mortality rate of breast cancer by 31%, and remains the most effective targeted cancer therapy. However, approximately one-third of patients treated with adjuvant tamoxifen suffer from aggressive recurrent disease. Resistance to tamoxifen, thus, remains a major challenge in providing effective treatments for these patients. In an effort to overcome the resistance, intensive research has been conducted to understand the underlying mechanisms; this has resulted in the identification of complex factors/pathways contributing to tamoxifen resistance, including modulations of the ERsignaling, upregulation of a set of growth factor receptor networks (HER2, EGFR, FGFR, and IGF1R), alterations of the PI3K-PTEN/AKT/mTOR pathway, and an elevation of the NF-κB signaling. Despite these advances, our understanding of the acquired resistance remains fragmented and there is a lack of a platform to integrate these diversified molecular factors/ pathways into a cohesive mechanistic model. Nonetheless, at the cellular level, it is becoming increasingly recongnized that cancer stem cells (CSCs) are key in driving cancer metastasis and therapy resistance. Likewise, evidence is emerging for the critical contributions of breast cancer stem cells (BCSCs) to tamoxifen resistance. In this review, we will discuss these recent developments of BCSC-mediated resistance to tamoxifen and the contributions of those demonstrated molecular factors/pathways to BCSC expansion during the emergency of tamoxifen resistance.