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Knockdown of HOTAIR inhibits EMT

Figure S1 depicts the location of CpG islands in promoter region of miR-182-5p

Prostate cancer incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to prostate cancer health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA prostate cancer. A comprehensive approach of using cell lines, tissue samples, and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR array, qRT-PCR, cell cycle, luciferase reporter, immunoblot, and IHC. Various statistical approaches such as Kaplan–Meier, uni-, and multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell-cycle pathway activation in AA patients with prostate cancer. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion, and induced cell-cycle arrest. Inhibition of miR-130b modulated critical prostate cancer–related biological pathways in AA compared with EA prostate cancer patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA prostate cancer. miR-130b is a significant contributor to prostate cancer racial disparity as its overexpression is a risk factor for poor prognosis in AA patients with prostate cancer. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of prostate cancer in AA patients.

Supplementary Figure 4. miR-130b cluster miRNA and MET expression level in TCGA PRAD samples.

Table S1 is for primer sequences

Supplemental Figure Legends

This study aims to investigate the role of miR-203–HOTAIR interaction in the suppression of renal cell carcinoma (RCC). We employed series of in vitro assays such as proliferation, invasion, migration, and colony formation along with in vivo tumor xenograft model. Profiling of miR-203 and HOTAIR expression revealed that miR-203 was significantly underexpressed, whereas HOTAIR was overexpressed in RCC cell lines and clinical specimens compared with normal cell line and tissue. Both miR-203 and HOTAIR expression significantly distinguished malignant from normal tissues and significantly correlated with clinicopathologic characteristics of patients. Overexpression of miR-203 significantly inhibited proliferation, migration, and invasion with an induction of apoptosis and cell-cycle arrest. However, HOTAIR suppression resulted in the similar functional effects in the same RCC cell lines. In silico, RNA-22 algorithm showed a binding site for miR-203 in HOTAIR. We observed a direct interaction between miR-203 and HOTAIR by RNA-immunoprecipitation (RIP) and luciferase reporter assays. We show that miR-203–HOTAIR interaction resulted in the inhibition of epithelial-to-mesenchymal transition (EMT) and metastatic genes as indicated by induction of key metastasis-suppressing proteins E-cadherin, claudin (epithelial markers), and PTEN along with induction of tumor suppressor genes p21 and p27. A significant decrease in vimentin (mesenchymal marker), KLF4, and Nanog (stemness markers) was also observed. This is the first report demonstrating miR-203–mediated regulation of HOTAIR induces tumor suppressor effects in RCC by regulating EMT and metastatic pathway genes. Thus, the study suggests that therapeutic regulation of HOTAIR by miR-203 overexpression may provide an opportunity to regulate RCC growth and metastasis. Mol Cancer Ther; 17(5); 1061–9. ©2018 AACR.

Antibody used with their catalog numbers

Primer Sequences

Supplementary figures S1-S6

Clinicopathological features

Supplementary Figures S1-2 and Tables S1-6. Supplementary Figure S1. Expression changes measured by qRT-PCR of genes undergoing DNA methylation shifts in canine epithelial-to-mesenchymal transition. Supplementary Figure S2. Translation of the DNA methylation changes observed in the canine epithelial-to-mesenchymal transition model to human breast cancer. Supplementary Table S1. List of all DMRs (87,296) identified between MDCK-U and MDCK-T. Supplementary Table S2. List of differentially expressed genes between MDCK-U and MDCK-T. Supplementary Table S3. Genes undergoing differential expression between MDCK-U and MDCK-T in association with an intragenic DMR. Supplementary Table S4. Gene ontology analysis of DNA methylation associated transcriptional changes in the MDCK-EMT model. Supplementary Table S5. Gene promoters with consistent DNA methylation variation in canine and human EMT. Supplementary Table S6. Gene ontology analysis with the gene-expression changes observed for conserved DMRs.

Although older people are at an increased risk of developing delirium during hospitalization, no definitive screening tools exist to predict the condition. This study aimed to examine the effectiveness of the noise pareidolia test (NPT) as a tool for predicting the onset of post-hospitalization delirium in older adults.Hospitalized patients who were cared for by a multidisciplinary geriatric care team owing to behavioral symptoms, difficulties in communication, and a history of dementia or delirium were analyzed. The NPT was performed on patients who could complete a Mini-Mental State Examination within 3 days of admission. Demographic and clinical data were recorded on the same day as the NPT or within 3 days of admission. Delirium was assessed using the observation-based Delirium Screening Tool (DST).Of 96 patients, 59 were in the DST-negative group and 37 in the DST-positive group. Benzodiazepine agonist use, serum potassium levels, and the number of images in which pareidolia was noted (i.e., the NPT score) significantly differed between groups. Logistic regression analysis identified benzodiazepine agonist use (odds ratio, 2.897; P = 0.032), serum potassium levels (odds ratio, 0.427; P = 0.041) and NPT scores (odds ratio, 1.253; P = 0.017) as significant predictors of DST results. The receiver operating characteristic curve analysis showed an NPT score of 1 as the appropriate cutoff value.A positive NPT score was identified as an independent predictor of delirium in older patients admitted to an acute care hospital with cognitive dysfunction. Geriatr Gerontol Int 2022; 22: 883-888.

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states.

Background Prostate cancer is one of the most commonly diagnosed cancers among men. African Americans (AA) are at an increased risk of developing prostate cancer compared to European Americans (EA). miRNAs play a critical role in these tumors, leading to tumor progression. In this study, we investigated the role of miR-182 in racial disparity in prostate cancer. Results We found significantly increased levels of miR-182 in prostate cancer tissues compared to BPH. Also, miR-182 shows increased expression in AA prostate cancer cell line and tissue samples compared to EA. We performed biochemical recurrence (BCR) - free survival time in AA and EA patients and found that high miR-182 expression had significantly shorter BCR-free survival than patients with low miR-182 expression (P = 0.031). To elucidate the role of miR-182, we knocked down miR-182 in EA (DU-145 and LNCaP) and AA (MDA-PCa-2b) cell lines and found an increase in apoptosis, arrest of the cell cycle, and inhibition of colony formation in the AA cell line to a greater extent than EA cell lines. Conclusions Our results showed that PDCD4 is a direct miR-182 target and its inhibition is associated with aggressiveness and high Gleason grade in prostate cancer among AA. These findings show that miR-182 is highly expressed in AA patients and miR-182 may be a target for effective therapy in AA patients.

Sex disparities in cardiac homeostasis and heart disease are well documented with male-female differences attributed to actions of sex specific hormones. However, recent studies implied sex chromosomes might act outside of the gonads to function in non-hormonal manner. How these two pathways contribute to cardiac sex differences remains unknown. Here, we performed both transcriptional and proteomics profiling to define the molecular differences between male and female mouse hearts. We demonstrate that, contrary to current dogma, cardiac sex disparities are controlled not solely by sex hormones, but also through a sex chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) mouse models, we find that the sex chromosome pathway is established by X-linked gene dosage. We also show that cardiac sex dipartites occur at the earliest stages of heart formation, a period before gonad formation. These studies provide new insights and resources for studying sex-biased cardiac disease states.

Protrudin is a protein that resides in the membrane of the endoplasmic reticulum and is highly expressed in the nervous system. Although mutations in the human protrudin gene (ZFYVE27, also known as SPG33) give rise to hereditary spastic paraplegia (HSP), the physiological role of the encoded protein has been largely unclear. We therefore generated mice deficient in protrudin and subjected them to a battery of behavioral tests designed to examine their intermediate phenotypes. The protrudin-deficient mice were found to have a reduced body size and to manifest pleiotropic behavioral abnormalities, including hyperactivity, depression-like behavior, and deficits in attention and fear-conditioning memory. They exhibited no signs of HSP, however, consistent with the notion that HSP-associated mutations of protrudin may elicit neural degeneration, not as a result of a loss of function, but rather as a result of a gain of toxic function. Overall, our results suggest that protrudin might play an indispensable role in normal neuronal development and behavior.

Male and female disease states differ in their prevalence, treatment responses, and survival rates. In cardiac disease, women almost uniformly fare far worse than men. Though sex plays a critical role in cardiac disease, the mechanisms underlying sex differences in cardiac homeostasis and disease remain unexplained. Here, in adult and embryonic hearts we reveal sex-specific transcriptomes and proteomes and show that cardiac sex differences are predominately accounted for by post-transcriptional mechanisms. We found differential expression of male-female proteins in the cardiomyocytes. Using a quantitative proteomics-based approach, we characterized differential sex-specific enriched cardiac proteins, protein complexes, and biological sex processes in the context of global genetic diversity of the Collaborative Cross, an established surrogate for human diversity. We also found that sex differences in cardiac protein expression are established by both hormonal and sex chromosomal mechanisms. We have demonstrated the onset of sex-biased protein expression and discovered that sex disparities in heart tissue occur at the earliest stages of heart development at a period that preceeds mammalian gonadal development. Collectively, these findings may explain why congenital heart disease, a leading cause of death whose origin is often developmental, is sex biased. Our results reveal molecular foundations for differences in cardiac tissue that underlie sex disparities in health, disease, and treatment outcomes.

The molecular heterogeneity of renal cell carcinoma (RCC) complicates the therapeutic interventions for advanced metastatic disease and thus its management remains a significant challenge. This study investigates the role of the lncRNA CDKN2B-AS1 and miR-141-3p interactions in the progression and metastasis of kidney cancer. Human renal cancer cell lines (ACHN and Caki1), normal RPTEC cells, tissue cohorts, and a series of in vitro assays and in vivo mouse model were used for this study. An overexpression of CDKN2B-AS1 was observed in RCC compared to normal samples in TCGA and our in-house SFVAMC tissue cohorts. Reciprocally, we observed reduced expression of miR-141 in RCC compared to normal in the same cohorts. CDKN2B-AS1 shares regulatory miR-141 binding sites with CCND1 and CCND2 genes. Direct interactions of CDKN2B-AS1/miR-141/Cyclin D1–D2 were confirmed by RNA immunoprecipitation and luciferase reporter assays indicating that CDKN2B-AS1/miR-141/Cyclin D1–D2 acts as a ceRNA network in RCC. Functionally, attenuation of CDKN2B-AS1 and/or overexpression of miR-141 inhibited proliferation, clonogenicity, migration/invasion, induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model. Further, overexpression of CDKN2B-AS1 is positively correlated with poor overall survival of RCC patients. Expression of miR-141 also robustly discriminated malignant from non-malignant tissues and its inhibition in normal RPTEC cells induced pro-cancerous characteristics. CDKN2B-AS1 attenuation or miR-141 overexpression decreased CCND1/CCND2 expression, resulting in reduced RAC1/pPXN that are involved in migration, invasion and epithelial–mesenchymal transition. This study, for the first time, deciphered the role of CDKN2B-AS1/miR-141/Cyclin D axis in RCC and highlights this network as a promising therapeutic target for the regulation of EMT driven metastasis in RCC.

Metastasis is the leading cause of mortality from kidney cancer, and understanding the underlying mechanism of this event will provide better strategies for its management. Here we investigated the biological, functional, and clinical significance of lncTCL6 and its interacting miR-155 in clear cell renal cell carcinoma (ccRCC). We employed a comprehensive approach to investigate the lncTCL6-miR-155-Src/Akt–mediated epithelial-to-mesenchymal transition (EMT) pathway as a novel regulatory mechanism in ccRCC progression. Expression analyses revealed that lncTCL6 is downregulated in ccRCC compared with normal tissues. Overexpression of lncTCL6 in ccRCC cell lines impaired their oncogenic functions, such as cell proliferation and migration/invasion, and induced cell-cycle arrest and apoptosis; conversely, depletion of lncTCL6 rescued these phenotypic effects. Furthermore, lncTCL6 directly interacted with miR-155. Unlike lncTCL6, miR-155 was overexpressed in ccRCC. Stable knockdown of miR-155 phenocopied the effects of lncTCL6 overexpression. Conversely, reconstitution of miR-155 and suppression of lncTCL6 in noncancerous renal cell HK2 induced tumorigenic characteristics. Patients with higher expression of lncTCL6 and lower expression of miR-155 had better survival probability. When overexpressed, lncTCL6 recruited STAU1 and mediated decay of Src mRNA, followed by a marked downregulation of an integrated network of Src target genes involved in migration, invasion, and EMT. However, the interaction between miR-155 and lncTCL6 attenuated the regulatory role of lncTCL6 on Src-mediated EMT. In conclusion, this study is the first report documenting the lncTCL6-miR155-Src/Akt/EMT network as a novel regulatory mechanism in aggressive ccRCC and a promising therapeutic target to inhibit renal cancer. Significance: This study's investigation of noncoding RNA interactions in renal cell carcinoma identify miRNA-155-lncRNA TCL6-mediated regulation of the Src-Akt-EMT network as a novel mechanism of disease progression and metastasis.

The co-evolution and co-existence of viral pathogens with their hosts for millions of years is reflected in dynamic virus-host protein-protein interactions (PPIs) that are intrinsic to the spread of infections. Here, we investigate the system-wide dynamics of protein complexes throughout infection with the herpesvirus, human cytomegalovirus (HCMV). Integrating thermal shift assays and mass spectrometry quantification with virology and microscopy, we monitor the temporal formation and dissociation of hundreds of functional protein complexes and the dynamics of host-host, virus-host, and virus-virus PPIs. We establish pro-viral roles for cellular protein complexes and translocating proteins. We show the HCMV receptor integrin beta 1 dissociates from extracellular matrix proteins, becoming internalized with CD63, which is necessary for virus production. Moreover, this approach facilitates characterization of essential viral proteins, such as pUL52. This study of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies., Here, Hashimoto et al. apply mass spectrometry-based thermal proximity coaggregation to characterize the temporal dynamics of virus-host protein-protein interactions during human cytomegalovirus (HCMV) infection, uncovering proviral functions including the internalization of the HCMV receptor integrin beta 1 with CD63.

Background/aims Genistein, a soy isoflavone, has been shown to have anti-cancer effects in various cancers including renal cancer. Long non-coding RNA, HOX transcript antisense RNA (HOTAIR), is involved in cancer progression and metastasis, such as renal cancer. Our aim was to investigate the effects of genistein on HOTAIR chromatin remodeling functions. Methods We used MTS assays and Transwell migration assays to study the effects of genistein on cell proliferation and migration respectively in human renal cell carcinoma (RCC) cell lines. We used Western blots to analyze SNAIL and ZO-1 expression. We performed chromatin immunoprecipitation (ChIP) assays to study recruitment of the polycomb repressive complex 2 (PRC2) to the ZO-1 promoter. We performed RNA immunoprecipitation (RIP) assays to study interaction between HOTAIR and PRC2, SMARCB1 or ARID1A. We also performed transfection experiments to overexpress EED, HOTAIR and knockdown SMARCB1. Results Genistein reduced cell proliferation and migration of human renal cell carcinoma cell lines. ChIP assays indicated that genistein reduces recruitment of the PRC2 to the ZO-1 promoter and increased its expression. RIP assays showed that genistein inhibits HOTAIR interaction with PRC2, leading to tumor suppression. Immunoprecipitation also revealed that genistein reduced EED levels in PRC2, suggesting that decreased EED levels suppress HOTAIR interaction with PRC2. EED overexpression in the presence of genistein restored PRC2 interaction with HOTAIR and reduced ZO-1 transcription, suggesting genistein activates ZO-1 by inhibiting HOTAIR/PRC2 functions. RIP assays also showed that HOTAIR interacts with SMARCB1 and ARID1A, subunits of the human SWI/SNF chromatin remodeling complex and genistein reduces this interaction. Combination of HOTAIR overexpression and SMARCB1 knockdown in the presence of genistein revealed that genistein inhibits SNAIL transcription via the HOTAIR/SMARCB1 pathway. Conclusion Genistein suppresses EED levels in PRC2 and inhibits HOTAIR/PRC2 interaction. Genistein suppresses HOTAIR/PRC2 recruitment to the ZO-1 promoter and enhances ZO-1 transcription. Genistein also inhibits SNAIL transcription via reducing HOTAIR/SMARCB1 interaction. We demonstrate that the reduction of HOTAIR interaction with chromatin remodeling factors by genistein represses HOTAIR/chromatin remodeling pathways to suppress RCC malignancy.

This study aims to investigate the role of miR-203–HOTAIR interaction in the suppression of renal cell carcinoma (RCC). We employed series of in vitro assays such as proliferation, invasion, migration, and colony formation along with in vivo tumor xenograft model. Profiling of miR-203 and HOTAIR expression revealed that miR-203 was significantly underexpressed, whereas HOTAIR was overexpressed in RCC cell lines and clinical specimens compared with normal cell line and tissue. Both miR-203 and HOTAIR expression significantly distinguished malignant from normal tissues and significantly correlated with clinicopathologic characteristics of patients. Overexpression of miR-203 significantly inhibited proliferation, migration, and invasion with an induction of apoptosis and cell-cycle arrest. However, HOTAIR suppression resulted in the similar functional effects in the same RCC cell lines. In silico, RNA-22 algorithm showed a binding site for miR-203 in HOTAIR. We observed a direct interaction between miR-203 and HOTAIR by RNA-immunoprecipitation (RIP) and luciferase reporter assays. We show that miR-203–HOTAIR interaction resulted in the inhibition of epithelial-to-mesenchymal transition (EMT) and metastatic genes as indicated by induction of key metastasis-suppressing proteins E-cadherin, claudin (epithelial markers), and PTEN along with induction of tumor suppressor genes p21 and p27. A significant decrease in vimentin (mesenchymal marker), KLF4, and Nanog (stemness markers) was also observed. This is the first report demonstrating miR-203–mediated regulation of HOTAIR induces tumor suppressor effects in RCC by regulating EMT and metastatic pathway genes. Thus, the study suggests that therapeutic regulation of HOTAIR by miR-203 overexpression may provide an opportunity to regulate RCC growth and metastasis. Mol Cancer Ther; 17(5); 1061–9. ©2018 AACR.

Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer.

The catechol-O-methylase (COMT) gene has been shown to play a protective role in the cell by reducing catechol-compounds that can cause genetic mutations and enhance the carcinogenesis process. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both The Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker or a therapeutic target for prostate cancer. Citation Format: Yuichiro Tanaka, Yutaka Hashimoto, Marisa Shiina, Varahram Shahryari, Priyanka Kulkarni, Soichiro Yamamura, Shahana Majid, Z Laura Tabatabai, Rajvir Dahiya. Functional properties and regulation of the catechol-O-methyltransferase gene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2501.

Understanding multicellular organism development from a molecular perspective is no small feat, yet this level of comprehension affords clinician-scientists the ability to identify root causes and mechanisms of congenital diseases. Inarguably, the maturation of molecular biology tools has significantly contributed to the identification of genetic loci that underlie normal and aberrant developmental programs. In combination with cell biology approaches, these tools have begun to elucidate the spatiotemporal expression and function of developmentally-regulated proteins. The emergence of quantitative mass spectrometry (MS) for biological applications has accelerated the pace at which these proteins can be functionally characterized, driving the construction of an increasingly detailed systems biology picture of developmental processes. Here, we review the quantitative MS-based proteomic technologies that have contributed significantly to understanding the role of proteome regulation in developmental processes. We provide a brief overview of these methodologies, focusing on their ability to provide precise and accurate proteome measurements. We then highlight the use of discovery-based and targeted mass spectrometry approaches in model systems to study cellular differentiation states, tissue phenotypes, and spatiotemporal subcellular organization. We also discuss the current application and future perspectives of MS proteomics to study PTM coordination and the role of protein complexes during development.

Understanding multicellular organism development from a molecular perspective is no small feat, yet this level of comprehension affords clinician-scientists the ability to identify root causes and mechanisms of congenital diseases. Inarguably, the maturation of molecular biology tools has significantly contributed to the identification of genetic loci that underlie normal and aberrant developmental programs. In combination with cell biology approaches, these tools have begun to elucidate the spatiotemporal expression and function of developmentally-regulated proteins. The emergence of quantitative mass spectrometry (MS) for biological applications has accelerated the pace at which these proteins can be functionally characterized, driving the construction of an increasingly detailed systems biology picture of developmental processes. Here, we review the quantitative MS-based proteomic technologies that have contributed significantly to understanding the role of proteome regulation in developmental processes. We provide a brief overview of these methodologies, focusing on their ability to provide precise and accurate proteome measurements. We then highlight the use of discovery-based and targeted mass spectrometry approaches in model systems to study cellular differentiation states, tissue phenotypes, and spatiotemporal subcellular organization. We also discuss the current application and future perspectives of MS proteomics to study PTM coordination and the role of protein complexes during development.

Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2′-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

Aberrant DNA methylation is frequently detected in gastrointestinal tumors, and can therefore potentially be used to screen, diagnose, prognosticate, and predict colorectal cancers (CRCs). Although colonoscopic screening remains the gold standard for CRC screening, this procedure is invasive, expensive, and suffers from poor patient compliance. Methylated DNA is an attractive choice for a biomarker substrate because CRCs harbor hundreds of aberrantly methylated genes. Furthermore, abundance in extracellular environments and resistance to degradation and enrichment in serum, stool, and other noninvasive bodily fluids, allows quantitative measurements of methylated DNA biomarkers. This article describes the most important studies that investigated the efficacy of serum- or stool-derived methylated DNA as population-based screening biomarkers in CRC, details several mechanisms and factors that control DNA methylation, describes a better use of prevailing technologies that discover novel DNA methylation biomarkers, and illustrates the diversity of demethylating agents and their applicability toward clinical impact.

This study investigated the role of the PI3K/Akt pathway in cadmium (Cd) induced malignant transformation of normal prostate epithelial (PWR1E and RWPE1) cells. Both PWR1E and RWPE1 cells were exposed to 10 μM Cd for one year and designated as Cd-PWR1E and Cd-RWPE1. Cd-RWPE1 cells robustly formed tumors in athymic nude mice. Functionally, Cd-exposure induced tumorigenic attributes indicated by increased wound healing, migration and invasion capabilities in both cell lines. RT2-array analysis revealed many oncogenes including P110α, Akt, mTOR, NFKB1 and RAF were induced whereas tumor suppressor (TS) genes were attenuated in Cd-RWPE1. This was validated by individual quantitative-real-time-PCR at transcriptional and by immunoblot at translational levels. These results were consistent in Cd-PWR1E vs parental PWR1E cells. Gene Set Enrichment Analysis revealed that five prostate cancer (PCa) related pathways were enriched in Cd-exposed cells compared to their normal controls. These pathways include the KEGG- Pathways in cancer, Prostate Cancer Pathway, ERBB, Apoptosis and MAPK pathways. We selected up- and down-regulated genes randomly from the PI3K/Akt pathway array and profiled these in the TCGA/GDC prostate-adenocarcinoma (PRAD) patient cohort. An upregulation of oncogenes and downregulation of TS genes was observed in PCa compared to their normal controls. Taken together, our study reveals that the PI3K/Akt signaling is one of the main molecular pathways involved in Cd-driven transformation of normal prostate epithelial cells to malignant form. Understanding the molecular mechanisms involved in the Cd-driven malignant transformation of normal prostate cells will provide a significant insight to develop better therapeutic strategies for Cd-induced prostate cancer.

Purpose Cadmium (Cd) is reported to be associated with carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer (PCa) remain elusive. Materials and methods RWPE1, PWR1E and DU 145 cells were used. RT2 Profiler Array, real-time-quantitative-PCR, immunofluorescence, cell cycle, apoptosis, proliferation and colony formation assays along with Gene Set Enrichment Analysis (GSEA) were performed. Result Chronic Cd exposure of non-malignant RWPE1 and PWR1E cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. Even a two-week Cd exposure of PCa cell line (DU 145) significantly increased the proliferation and decreased apoptosis. RT2 profiler array of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis in both Cd-RWPE1 and Cd-PWR1E cell lines. GSEA showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from Erk/MAPK pathway and performed profile analysis in a PCa data set from the TCGA/GDC data base. We observed upregulation of these genes in PCa compared to normal samples. An increase in phosphorylation of the Erk1/2 and Mek1/2 was observed in Cd-RWPE1 and Cd-PWR1E cells compared to parental cells, confirming that Cd-exposure induces activation of the Erk/MAPK pathway. Conclusion This study demonstrates that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate cells. Understanding these dominant oncogenic pathways may help develop optimal therapeutic strategies for PCa.

Prostate cancer (PC) incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to PC health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA PC. A comprehensive approach of using cell lines, tissue samples and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR-array, qRT-PCR, cell cycle, luciferase reporter, immunoblot and immunohistochemistry. Various statistical approaches such as Kaplan-Meier, Uni- and Multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell cycle pathway activation in AA PC patients. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion and induced cell cycle arrest. Inhibition of miR-130b modulated critical PC related biological pathways in AA compared to EA PC patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA PC. miR-130b is a significant contributor to PC racial disparity as its overexpression is a risk factor for poor prognosis in AA PC patients. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of PC in AA patients. Citation Format: Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Rajvir Dahiya, Shahana Majid. Upregulation of miR-130b contributes to risk of poor prognosis and racial disparity in African-American prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B069.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5′ untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G‐C‐T‐G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non‐smokers and non‐drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G‐C‐T‐G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype.

The molecular heterogeneity of clear cell renal carcinoma (ccRCC) makes prediction of disease progression and therapeutic response difficult. Thus, this report investigates the functional significance, mechanisms of action, and clinical utility of miR-182-5p and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1/NEAT2), a long noncoding RNA (lncRNA), in the regulation of kidney cancer using human kidney cancer tissues as well as in vitro and in vivo model systems. Profiling of miR-182-5p and MALAT-1 in human renal cancer cells and clinical specimens was done by quantitative real-time PCR (qPCR). The biological significance was determined by series of in vitro and in vivo experiments. The interaction between miR-182-5p and MALAT-1 was investigated using luciferase reporter assays. In addition, the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell-cycle progression were assessed in ccRCC cells. The data indicate that miR-182-5p is downregulated in ccRCC; the mechanism being CpG hypermethylation as observed from 5-Aza CdR treatment that decreased promoter methylation and expression of key methylation regulatory genes like DNMT1, DNMT3a, and DNMT3b. Overexpression of miR-182-5p–inhibited cell proliferation, colony formation, apoptosis, and led to G2–M-phase cell-cycle arrest by directly targeting MALAT-1. Downregulation of MALAT-1 led to upregulation of p53, downregulation of CDC20, AURKA, drivers of the cell-cycle mitotic phase. Transient knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Finally, overexpression of miR-182-5p decreased tumor growth in mice, compared with controls; thus, demonstrating its antitumor effect in vivo. Implications: This is the first study that offers new insight into role of miR-182-5p/MALAT-1 interaction on inhibition of ccRCC progression. Mol Cancer Res; 16(11); 1750–60. ©2018 AACR.

Summary Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading to increased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.

Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) responds to growth factors and nutrient availability. Amino acids induce the recruitment of mTORC1 to the lysosomal membrane and its consequent activation, but the molecular mechanism of such activation has remained unclear. We have now examined the role of TMEM55B, a lysosomal protein of unknown molecular function, in this process on the basis of the results of proteomics and immunofluorescence analyses showing that TMEM55B interacts with many proteins that participate in mTORC1 activation including components of the vacuolar-type proton ATPase (V-ATPase) and Ragulator complexes at the lysosomal membrane. The amino acid-induced phosphorylation of the mTORC1 substrates S6K and 4E-BP was attenuated in TMEM55B-depleted cells compared with control cells. Depletion of TMEM55B was also found to evoke lysosomal stress as showed by translocation of the transcription factor TFEB to the nucleus. Furthermore, recruitment of the V1 domain subcomplex of V-ATPase to lipid rafts was abrogated in TMEM55B-depleted cells. Collectively, our results suggest that TMEM55B contributes to assembly of the V-ATPase complex in lipid rafts of the lysosomal membrane and to subsequent activation of mTORC1.

The most frequent alteration in the prostate oncogenome is loss of chromosome (chr) 8p21 that has been associated with loss of NKX3.1 homeobox gene. Chr8p21 deletions increase significantly with tumor grade and are associated with poor prognosis in prostate cancer (PCa), suggesting critical involvement of this region in tumor progression. Recent studies suggest that apart from NKX3.1, this region harbors alternative tumor suppressors that are yet undefined. We proposed a novel, paradigm shifting hypothesis that this locus is associated with a miRNA gene cluster-miR-3622a/b- that plays a crucial suppressive role in PCa. Here we demonstrate the crucial role of miR-3622a in prostate cancer epithelial-to-mesenchymal transition (EMT). MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a expression is widely downregulated and is significantly correlated with poor survival outcome and tumor progression. To understand the functional significance of miR-3622a, knockdown and overexpression was performed using non-transformed prostate epithelial and PCa cell lines, respectively, followed by functional assays. Our data demonstrate that endogenous miR-3622a expression is vital to maintain the epithelial state of normal and untransformed prostate cells. miR-3622a expression inhibits EMT, progression and metastasis of PCa in vitro and in vivo. Further, we found that miR-3622a directly targets EMT effectors ZEB1 and SNAI2. In view of these data, we propose that frequent loss of miR-3622a at chr8p21 region leads to induction of EMT states that in turn, promotes PCa progression and metastasis. This study has potentially significant implications in the field of prostate cancer as it identifies an important miRNA component of a frequently lost chromosomal region with critical roles in prostate carcinogenesis which is a highly significant step towards understanding the mechanistic involvement of this locus. Also, our study indicates that miR-3622a is a novel PCa biomarker and potential drug target for developing therapeutic regimens against advanced PCa.