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Social determinants of health influence health outcomes and life expectancy. Specifically, individuals living in poverty often have adverse health outcomes related to chronic inflammation that affect the cardiovascular, renal, and pulmonary systems. However, the underlying mechanisms by which poverty increases the risk of disease and health disparities are still not fully understood. To bridge the gap in our understanding of the link between living in poverty and adverse health outcomes, we performed RNA sequencing of blood immune cells from 204 participants of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study in Baltimore City, Maryland. This study cohort included men and women self-identified as African American and White. We identified 581 genes differentially expressed in association with poverty. A larger number of differentially expressed genes were detected in women, compared to men, and 64 genes had distinct sex-by-poverty interaction effects. Genes differentially expressed in women living in poverty were enriched in wound healing and coagulation processes, while in men were mostly related to immunoglobulin production and humoral immune response. Of the genes differentially expressed in individuals living in poverty, 275 are also associated with complex diseases in transcriptome-wide association studies. Our results suggest that living in poverty influences inflammation and the risk for chronic disease through gene expression changes in immune cells, and that some of the effects of living in poverty are different in women and men.

Research partnerships between universities and communities following the principles of community-based participatory research (CBPR) have the potential to eliminate cycles of health disparities. The purpose of this article is to describe the process of establishing a community-campus network with a distinct mission and vision of developing trusting and successful research partnerships that are sustained and effective. In 2019, Morgan CARES was established to facilitate community engagement by founding a community center “within” a low-income residential neighborhood as a safe and accessible hub for creating a vibrant learning community. A community needs assessment and asset mapping was conducted and several necessary resources and services were provided to maximize networking opportunities, nurture innovative ideas and proposals, and provide seed funding. Lessons learned informed the optimization of a theoretical model that has guided the development and implementation of the program’s key components. By December 2021, Morgan CARES had recruited 222 community and 137 academic members representing diverse expertise from across Baltimore City. We also successfully established new partnerships and funded a total of 17 small community-academic awards. Although in its early stages, Morgan CARES has established a dynamic learning community following a conceptual framework that could guide future similar initiatives.

Prostate cancer is one of the common types of cancer and remains the second leading cause of cancer-related deaths in men in the United States. High Mobility Group Protein AT-hook 2 (HMGA2), a DNA binding protein acts as a transcriptional regulating factor in gene transcription and facilitates epithelial-mesenchymal transition (EMT), which is usually the onset of prostate cancer progression and metastasis. HMGA2 isoforms include wild-type or full length that induces EMT, while the truncated isoform is associated with elevated proliferation and migration. Enzalutamide is a second-generation antiandrogen drug known to inhibit androgen receptor (AR) translocation into the nucleus by competitively binding AR and preventing androgen binding. Alisertib (MLN8237) is a small molecule inhibitor of Aurora kinase A has been utilized in clinical trials for neuroendocrine cancers and can also inhibit EMT. We aim to investigate which of the two drugs is effective in reducing proliferation in prostate cells overexpressing HMGA2 isoforms (wild-type and truncated). In this study, we treated LNCaP cells overexpressing HMGA2 with enzalutamide (1-30 µM) and alisertib (2.5-40 µM). Treatment with enzalutamide and alisertib indicated a dose-dependent decrease in cell proliferation with optimal dose at 20 µM for both drugs. LNCaP cells (LNCaP Neo) expressing low level of HMGA2 were observed to respond better to enzalutamide treatment, while those overexpressing HMGA2 (WT and TR) respond better to alisertib treatment. Interestingly, treatment with 20 µM enzalutamide and alisertib followed by Western blot indicated enzalutamide to have no effects on protein expression of HMGA2 isoforms, but greatly decreased the expression of AR in LNCaP cell overexpressing truncated HMGA2. On the other hand, alisertib increased the expression of wild-type HMGA2 whilst having no effects on protein expression of AR. Both drugs at 20 µM did not have any significant effect on the expression of EMT markers (snail and vimentin).In conclusion, alisertib is more potent than enzalutamide in decreasing cell proliferation in prostate cancer cells expressing HMGA2. Further studies need to be conducted to understand the role of HMGA2 in promoting resistance to enzalutamide and whether alisertib may be a better drug for patients that express HMGA2. Acknowledgements: These studies were supported by NIH/NIMHD 2U54MD007590 and 5U54MD013376-8281. Citation Format: Yusuf Mansur Liadi, Valerie Odero-Marah. Antiproliferative activity of enzalutamide and alisertib in prostate cancer cells overexpressing HMGA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1572.

Prostate cancer (PCa) is the most common non-skin cancer in men, and second leading cause of death. PCa occurs when cells within the prostate grow uncontrollably, creating small tumors. Generally, the growth/proliferation of the prostate is regulated by the male hormone, androgen, that binds androgen receptor (AR), a nuclear receptor that functions as a transcription factor and regulates the development and proliferation of the prostate as well as PCa. Interestingly, AR has become one of the leading pathways targets to slow down prostate cancer progression. In fact, enzalutamide a second-generation antiandrogen drug works to inhibit AR translocation into the nucleus. High Mobility Group AT-Hook (HMGA2) is a transcription factor protein that alters the structure of DNA by binding to the promoter region to regulate the transcription of genes. The expression of HMGA2 is associated with cancer proliferation and metastasis and is higher in PCa patients compared to men without cancer. Additionally, this gene has two isoforms HMGA2 full-length/wild-type and truncated (missing the carboxyl terminus). Preliminary studies shown that AR is localized in the nucleus of HMGA2 wild-type but not truncated overexpressing cells. Our hypothesis is that HMGA2 wild-type promotes AR translocation to the nucleus which leads to increased cell proliferation in PCa cells. We utilized HMGA2-wild-type and HMGA2-truncated overexpressing LNCaP cells and C4-2B enzalutamide resistant cells to knockdown either AR or HMGA2 with siRNA followed by western blot analysis, cell proliferation assay, and immunofluorescence. Our results show that HMGA2 siRNA led to decreased expression of wild-type (25 kDa long form) and truncated isoform (17 kDa) protein expression, and cell proliferation when compared to control siRNA. However, AR knockdown decreased AR expression in both cell lines but only decreased cell proliferation in HMGA2-wild-type and C4-2B enzalutamide-resistant cells. This suggests that HMGA2 wild-type works with AR to promote cell proliferation, whereas HMGA2-truncated promotes cell proliferation by an AR-independent mechanism. Therefore, we need to examine how current PCa treatments such as enzalutamide that target AR work in patients expressing differing HMGA2 isoforms. Grant/Other Support: RISE: 5R25GM0058904, NIH/NIMHD 2U54MD007590; 5U54MD013376-8281; NSF S-STEM 2030608. Citation Format: Mojisoluwa Simisoluwa Awolowo, Taaliah Campbell, Ohuod Hawsawi, Valerie Odero-Marah. Investigating the role of HMGA2 and AR crosstalk in prostate cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 340.

Prostate cancer is the most common non-cutaneous cancer among men. A man with 1,2 or 3 first degree relatives with prostate cancer, has a 2,5, and 11-fold increased risk of developing prostate cancer. The heritability rate of prostate cancer is 58%. African American men have the highest incidence and mortality rate of prostate cancer in American. Men of African descent, globally, are more likely to die from prostate cancer than any other ancestral groups. We performed Immunohistochemistry(IHC) and RNA seq analysis on African American tissue and serum samples, with a focus on HMGA2, which has been shown promote EMT, invasion, and metastasis in cancer. Acknowledgements: These studies were supported by NIH/NIMHD U54MD007590 and U54MD013376. Our sample set included 3 benign and 2 prostate cancer tumors from African Americans. IHC markers showed HMGA2 staining within epithelial cells of the prostate cancer tissue. This result validates studies that show that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types. RNA seq results showed HMGA to be non-significantly down-regulated in tumor samples. The most differentially expressed (DE) gene, however, was SNORD116-18. Studies have shown SNORD116-18 expression to be associated with chronic lymphocytic leukemia in distinguishing prognostic groups. This finding may be a pathway of interest for further studies. RNA seq of serum gave homogeneous results, however, gene set enrichment analysis of the most differentially expressed genes within the serum showed overlap with inflammation pathways gene for upregulated DE genes and Methylation Pathway for downregulated DE genes. Further analysis on more samples from African Americans tissue and serum will be used to validate findings. Citation Format: Maxine S. Harlemon, Roni Bollag, Martha Terris, Ana Cecilia Millena, Nathan Bowen, Valerie Odero-Marah. A case and control genetic profile of tissue and serum in African American men [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C044.

Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men. Compared to Caucasian men, African American males have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that High Mobility Group A2 (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions. We have previously published that PCa cells degrades hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that HMGA2 isoforms may play differential roles at the bone metastatic site and effect paracrine cell proliferation and migration. LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite or human bone matrix powder, for 6 days followed by collection of conditioned media (CM). CM was co-cultured with parental LNCaP cells for various time points followed by analysis of several signaling pathways using western blot analysis, as well as analysis of paracrine cell proliferation using MTS assay, and cell migration across collagen using boyden chamber. Results reveal that co-culture of LNCaP cells expressing WT and TR HMGA2 with bone components has a paracrine effect on MAPK signaling. This was accompanied by a paracrine increase in cell migration but not cell proliferation with CM from truncated HMGA2-expressing cells co-cultured with bone, that can be abrogated by MAPK inhibition. Our study shows that wild-type and truncated isoforms of HMGA2 may differentially mediate PCa/hydroxyapatite bone interactions at the bone metastatic site, which may contribute to bone metastasis, particularly in African American men that have higher bone density. Acknowledgements: These studies were supported by NIH/NIGMS/RISE SR25GM060414 and NIH/NIMHD 2U54MD007590; 5U54MD013376-8281. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Veronica Henderson, Valerie Odero-Marah. Investigating the differential roles of HMGA2 isoforms in the bone microenvironment [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C033.

The aryl hydrocarbon receptor (AhR) is a nuclear transcription factor and xenobiotic sensor reported to mediate diet-induced obesity and is upregulated in high-grade prostate cancer (PCa). Previously published data from our lab shows AhR protein is overexpressed and constitutively active in castration-resistant PCa cells. AhR signaling is promoted by high fat diet-induced obesity, when circulating leptin levels are also high. Physiologically relevant leptin concentration is proportional to BMI and is secreted by adipose cells to inhibit hunger under normal circumstances. Our preliminary results suggest that both AhR and leptin independently desensitize PCa cells to clinically-relevant chemotherapeutics however, the underlying mechanisms must be elucidated and may require sustained AhR signaling. We also conducted an integrative bioinformatics analysis to explore the role of AHR and LEPR in PCa, which revealed AHR and LEPR mRNA expression positively correlates in prostate cancer (P Citation Format: Kofi K. Khamit-Kush, Joann B. Powell, Jeffrey A. Handy, Nathan J. Bowen, Valerie Odero-Marah, Daqing Wu. Aryl hydrocarbon receptor and leptin promote prostate cancer progression and may be associated with chemoresistance [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C023.

Prostate cancer (PCa) and breast cancer (BCa) are leading causes of cancer related deaths in American men and women, and morbidity is due to metastasis preferentially to bone. Compared to Caucasian American (CA), African American (AA) men and women have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that high mobility group two (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting epithelial-mesenchymal transition (EMT) and metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions, particularly, how PCa cells interact with bone at the metastatic site. We have previously published that PCa cells may degrade hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that EMT is associated with prostate health disparities and that HMGA2 isoforms may play differential roles at the bone metastatic site. We examined EMT marker expression in breast and prostate patient tissue and cell lines by immunohistochemistry (IHC), RNA In Situ Hybridization (RISH), western blot, and real-time PCR. Next, LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite, demineralized bone matrix or human ground bone matrix, for 6 days followed by collection of condition media (CM). CM was added to parental LNCaP cells for various time points followed by analysis of paracrine cell proliferation using MTS assay, cell migration across collagen using boyden chamber, and the effect on various pathways using western blot analysis. EMT marker (Snail and Cathepsin L) protein expression was higher in AA normal and cancer tissue compared to CA, while it was intermediate in Bahamian tissue. Wild-type HMGA2 mRNA expression was higher in metastatic patient tissue compared to truncated HMGA2, and higher in AA compared to CA tissue. CM taken from LNCaP cells overexpressing wild-type and truncated HMGA2 co-cultured with hydroxyapatite, demineralized bone matrix or ground bone matrix when added to parental LNCaP cells led to higher paracrine cell proliferation and paracrine cell migration. This was associated with MAPK activation that could be abrogated by MAPK inhibitor, UO126. These results indicate that EMT is higher in AA compared to CA. Additionally, both wild-type and truncated HMGA2 interaction with bone matrix promotes paracrine cell proliferation and migration via ERK signaling. Our study shows that wild-type and truncated isoforms of HMGA2 mediate PCa/bone interactions which may lead to increased bone metastasis, particularly in African American men that have higher bone density. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Bor-Jang Hwang, Liza J. Burton, Quentin Loyd, Veronica Henderson, Simone Howard, Camille Ragine, Robin Roberts, Andrew Gachii, Valerie Odero-Marah. Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA041.

People of African descent suffer more from cancer compared to other racial groups in terms of morbidity and mortality. Surveillance, Epidemiology, and End Results (SEER) reports Black men and women to have the highest diagnoses and highest cancer death rate. The reported cancer disparity statistic is also reflected by cancer type; prostate cancer was shown to be one of the top causes of cancer death in all cancer types, and the second leading cause of death, after lung cancer, in men. Men of African Descent, however, have more than a 2-fold increased mortality rate of prostate cancer compared to other ancestral populations. Cancer is considered a genetic disease, and prostate cancer has a heritability of 58%. Most studies however, do not capture the genetic heterogeneity found in men of African Descent, nor histological and morphological profiles that would provide prognostic and diagnostic markers for targeted and personalized therapeutic treatment options. Visium Spatial Transcriptomics is a method that maps transcriptomics data to the location within the tissue region that is being analyzed. Instead of being limited to a small piece of DNA or RNA, spatial transcriptomics enables high-resolution assessment of spatial gene expression across tissue sections. This technology assiduously probes the transcriptome of tissues samples in an untargeted way. Spatial Transcriptomics involves cutting edge technology that provides great potential to capture the cellular landscape of men with the heaviest burden of prostate cancer. The Visium platform by 10X Genomics is able to interrogate more than 10,000 transcripts per capture region. The protocol involves a comprehensive process from tissue collection to next generation sequencing following a bioinformatics pipeline. The complete protocol from beginning to end requires a collaboration of clinicians, pathologists, basic scientists, and bioinformaticians. This means that access to resources is imperative to the success of this technique. Unfortunately, low resource institutions are at a disadvantage in being able to perform the entire protocol. This fact is yet another type of health disparity. This observation, however, provides great opportunities to establish collaborations with other institutions. Closing the gap between higher and lower resource investigations would be vital in addressing prostate cancer health disparity using spatial transcriptomics. I will share my observation, learning, experimentation, and joint participation effort experience with Visium Spatial Transcriptomics in my quest to understand the genetic architecture of men of African descent that has them at a prolific risk for prostate cancer. Citation Format: Maxine S. Harlemon, Ana C. Millena, Bor-Jang Hwang, Valerie Odero-Marah. Utilizing Visium spatial transcriptomics to investigate prostate cancer disparity in men of African descent [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA025.

High mobility group A2 (HMGA2), a non-histone protein, is known to promote epithelial-mesenchymal transition (EMT), which plays a critical role in prostate cancer progression and metastasis. Both full-length HMGA2 (WT-HMGA2) and truncated (lacking the 3’UTR) HMGA2 (TR-HMGA2) isoforms are overexpressed in several cancers such as lung carcinomas, ovarian cancer, breast cancer, and gastric cancer. However, there is no study investigating the expression and differential roles of WT vs truncated HMGA2 isoforms in prostate cancer. Our previous results indicated that the overexpression of HMGA2 in LNCaP cells increased cell viability and migration for both wild-type and truncated HMGA2. Promotion of EMT was observed in wild-type, but not truncated HMGA2. Reactive oxygen species (ROS) levels were increased with truncated HMGA2 more than wild-type HMGA2. Therefore, wild-type and truncated HMGA2 may play different roles on cancer progression and metastasis. The goal of this study is to examine the expression of wild-type vs. truncated HMGA2 in prostate cancer patient tissue. We have developed a way to detect location and expression of wild-type and truncated HMGA2 using RNA in situ hybridization (RISH). The specific probes’ hybridization followed by a serial signal amplification process allows us to locate and quantify the HMGA2 isoforms in Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from cancer patients. Several prostate tissue samples and a prostate cancer tissue microarray that includes 35 patients from different races, stages of cancer, and metastasis status was analyzed. Our results showed increased HMGA2 expression (both wild-type and truncated) in several prostate cancer samples with higher stage cancer. We also observed that most wild-type and truncated HMGA2 is located within the nucleus, while some wild-type isoforms were detected within the cytoplasm. We will further quantify the expression levels to examine whether they are associated with tumor stage and/or race. These studies are the first to examine HMGA2 isoform expression and localization in prostate patient tissue and may offer novel therapeutic intervention strategies based on HMGA2 isoform expression. Citation Format: Bor-Jang Hwang, Mojisoluwa Awolowo, Taaliah Campbell, Ohuod Hawsawi, Sharon Harrison, Denise Gibbs, Camille Ragin, Valerie Odero-Marah. Detection of wild-type and truncated HMGA2 in prostate cancer tissues using RNA in situ hybridization [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C017.

Prostate cancer (PCa) patients' mortality is mainly attributed to complications caused by metastasis of the tumor cells to organs critical for survival, such as bone. We hypothesized that PCa cell-bone interactions would promote paracrine signaling. A panel of PCa cell lines were cocultured with hydroxyapatite ([HA]; inorganic component of bone) of different densities. Conditioned media (CM) was collected and analyzed for calcium levels and effect on paracrine signaling, cell migration, and viability in vitro and in vivo. Our results showed that calcium levels were elevated in CM from cancer cell-bone cocultures, compared to media or cancer cells alone, and this could be antagonized by ethylene glycol-bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator, or knockdown of Snail protein. We also observed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation and paracrine cell proliferation and migration in LNCaP cells incubated with CM from various cell lines; this phosphorylation and cell migration could be antagonized by Snail knockdown or various inhibitors including EGTA, STAT3 inhibitor (WP1066) or cathepsin L inhibitor (Z-FY-CHO). In vivo, higher HA bone density increased tumorigenicity and migration of tumor cells to HA implant. Our study shows that cancer-bone microenvironment interactions lead to calcium-STAT3 signaling, which may present an area for therapeutic targeting of metastatic PCa.

Prostate cancer (PCa) is one of the most commonly diagnosed malignancies among men worldwide and remains the second leading cause of cancer related death in the United States. Oxidative stress has been shown to be increase in several cancers including prostate cancer. In fact, oxidative stress in prostate cancer is suggested to be a direct result of cell exposure to reactive oxygen species (ROS). High mobility group A 2 (HMGA2), a non-histone protein, is an oncogene that is up-regulated in several cancers. This protein has ability to undergo chromosomal rearrangement and alternative splicing, causing its full length/wild type HMGA2 (HMGA2-WT) to become the truncated losing its 3’UTR leading to the generation of HMGA2 truncated (HMGA2-TR). We have previously shown HMGA2-WT’s involvement in epithelial mesenchymal transition (EMT), however, the functional role of HMGA2-TR has not yet been dissected. We hypothesize that truncated HMGA2’s involvement with oxidative stress leads to prostate cancer progression. We analyzed the baseline expression of wild-type vs.truncated HMGA2 in prostate patient tissue and cells lines by real-time PCR and western blot analyses. Prostate cancer patient tissue and some cell lines expressed increasing amounts of both wild-type and truncated HMGA2with increasing tumor grade, when compared to normal epithelial cells. RNA-Seq analysis of LNCaP prostate cancer cells stably overexpressing HMGA2-WT, HMGA2-TR, or empty vector (Neo) control revealed thatHMGA2-TR cells display increased oxidative stress compared to HMGA2-WT or Neo control cells. This was also confirmed by analysis of basal reactive oxygen species (ROS) levels, and the ratio of GSH/GSSG andNADP/NADPH utilizing metabolomics. Additionally, proteomic analysis showed that HMGA2-TR protein interacted with several proteins, including a cytoplasmic stress granule protein G3BP1 that responds to oxidative stress. Transient knockdown of G3BP1 increased ROS in HMGA2-TR cells. These studies may therefore uncover novel role for truncated HMGA2 in oxidative stress. Acknowledgements: These studies were supported by the NIH/NIMHD/RCI Grant #5G12MD007590-31,NIH/NIGMS/RISE Grant #5R25GM060414 Citation Format: Taaliah Campbell, Ohuod Hawsawi, Nathan Bowen, Valerie Odero-Marah. Investigating the role of high mobility group a2 (HMGA2) truncated isoform in promoting oxidative stress in PCa cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 113.

The aryl hydrocarbon receptor (AhR) is a nuclear transcription factor and xenobiotic sensor reported to mediate diet-induced obesity and is upregulated in high-grade prostate cancer (PCa). Previously published data from our lab shows that AhR protein is overexpressed and constitutively active in castration-resistant prostate cancer cells. Physiologically relevant leptin concentration is proportional to BMI and it is secreted by adipose cells to inhibit hunger under normal circumstances. Our preliminary results suggest that obesity-associated leptin concentrations desensitize PCa cells to cancer drugs however, the underlying mechanisms must be elucidated and may require sustained AhR signaling. In this study, we conducted an integrative bioinformatics analysis to explore the role of AHR and LEPR in PCa, which revealed AHR and LEPR mRNA expression positively correlates in prostate cancer (P Citation Format: Kofi Kyenku Khamit-Kush, Joann B. Powell, Jeffrey A. Handy, Nathan Bowen, Daqing Wu, Valerie Odero-Marah. Elevated aryl hydrocarbon receptor and leptin receptor expression correlates with prostate cancer progression and may be associated with obesity-associated leptin-mediated chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 394.

African American men (AA) suffer disproportionately from prostate cancer (PCa) displaying higher incidence and mortality rates when compared to Caucasian-American men (CA). Recent studies have shown that high mobility group A2 (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting metastasis. HMGA2 full-length/wild-type and truncated (lacking the 3'UTR) isoforms are overexpressed in several cancers, however, their distinct roles in PCa have not been reported. Literature has previously revealed a splicing factor, CDC-like kinase 3 (CLK3), that promotes splicing of wild-type HMGA2 to create the truncated HMGA2 isoform. We hypothesize that HMGA2 isoforms may play differential roles to promote PCa progression according to ethnicity. qPCR analysis of patient tissue of varying PCa stages was conducted to look at the mRNA expression of HMGA2 isoforms and CLK3. RNA-Seq was performed on LNCaP prostate cancer cell lines stably overexpressing wild-type or truncated HMGA2 and compared to Neo control. Both wild-type and truncated HMGA2 increased with increased PCa gleason grade. AA when compared to CA, displayed higher levels of truncated HMGA2 and its splicing factor, CLK3. RNA-Seq analysis revealed distinct gene sets regulated by truncated HMGA2 compared to wild-type HMGA2, including upregulation of a distinct set of genes involved in androgen signaling. These results indicate that HMGA2 isoforms are disproportionately expressed in AA and CA and increase with PCa progression. The regulation of differential genes by HMGA2 isoforms suggest that they may use distinct pathways to promote PCa. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Valerie Odero-Marah. The role of HMGA2 and CLK3 in prostate cancer health disparities [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR-17.

Inadequate nutrient intake leads to oxidative stress disrupting homeostasis, activating signaling, and altering metabolism. Oxidative stress serves as a hallmark in developing prostate lesions, and an aggressive cancer phenotype activating mechanisms allowing cancer cells to adapt and survive. It is unclear how adaptation and survival are facilitated; however, literature across several organisms demonstrates that a reversible cellular growth arrest and the transcription factor, nuclear factor-kappaB (NF-κB), contribute to cancer cell survival and therapeutic resistance under oxidative stress. We examined adaptability and survival to oxidative stress following nutrient deprivation in three prostate cancer models displaying varying degrees of tumorigenicity. We observed that reducing serum (starved) induced reactive oxygen species which provided an early oxidative stress environment and allowed cells to confer adaptability to increased oxidative stress (H2O2). Measurement of cell viability demonstrated a low death profile in stressed cells (starved + H2O2), while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no significant cell death in stressed cells suggesting an adaptive mechanism to tolerate oxidative stress. Stressed cells also presented a quiescent phenotype, correlating with NF-κB nuclear translocation, suggesting a mechanism of tolerance. Our data suggests that nutrient deprivation primes prostate cancer cells for adaptability to oxidative stress and/or a general survival mechanism to anti-tumorigenic agents.

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

The sixth International African–Caribbean Cancer Consortium (AC3) Conference was held 6–9 October 2017 in Miami, Florida, U.S.A. The conference was open to all researchers, trainees, clinical and public health professionals, and community members, and served as an international hub for the United States, the Caribbean, and Africa. Sessions included AC3 collaboration meetings, cancer surveillance and research skills training workshops, and a community cancer prevention conference.

Creosote bush (Larrea tridentata, LT) leaves extracts were tested for their potential efficacy to mitigate cellular oxidative stress on human SH-SY5Y cells. Here, the differential nuclear staining assay, a bioimager system, and flow cytometric protocols, concurrently with several specific chemicals, were used to measure the percentage of cell viability and several facets implicated in the cytoprotective mechanism of LT extracts. Initially, three LT extracts, prepared with different solvents, ethanol, ethanol:water (e/w), and water, were tested for their capacity to rescue the viability of cells undergoing aggressive H2O2-induced oxidative stress. Results indicate that the LT extract prepared with a mixture of ethanol:water (LT-e/w, 60:40% v/v) displayed the most effective cytoprotection rescue activity. Interestingly, by investigating the LT-e/w mechanism of action, it was found that LT-e/w extract decreases the levels of H2O2-provoked reactive oxidative species (ROS) accumulation, mitochondrial depolarization, phosphatidylserine externalization, caspase-3/7 activation, and poly (ADP-ribose) polymerase (PARP) cleavage significantly, which are hallmarks of apoptosis. Thus, out of the three LT extracts tested, our findings highlight that the LT-e/w extract was the most effective protective reagent on SH-SY5Y cells undergoing oxidative stress in vitro, functioning as a natural anti-apoptotic extract. These findings warrant further LT-e/w extract examination in a holistic context.

Typically the normal epithelial cells are a single layer, held tightly by adherent proteins that prevent the mobilization of the cells from the monolayer sheet. During prostate cancer progression, the epithelial cells can undergo epithelial-mesenchymal transition or EMT, characterized by morphological changes in their phenotype from cuboidal to spindle-shaped. This is associated with biochemical changes in which epithelial cell markers such as E-cadherin and occludins are down-regulated, which leads to loss of cell-cell adhesion, while mesenchymal markers such as vimentin and N-cadherin are up-regulated, thereby allowing the cells to migrate or metastasize to different organs. The EMT transition can be regulated directly and indirectly by multiple molecular mechanisms including growth factors and cytokines such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF) and insulin-like growth factor (IGF), and signaling pathways such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K). This signaling subsequently induces expression of various transcription factors like Snail, Twist, Zeb1/2, that are also known as master regulators of EMT. Various markers associated with EMT have been reported in prostate cancer patient tissue as well as a possible association with health disparities. There has been consideration to therapeutically target EMT in prostate cancer patients by targeting the EMT signaling pathways.

Studies have shown that High mobility group A2 (HMGA2), a non-histone protein, can promote epithelial-mesenchymal transition (EMT), which plays a critical role in prostate cancer progression and metastasis. Interestingly, full-length or wild-type HMGA2 and truncated (lacking the 3'UTR) HMGA2 isoforms are overexpressed in several cancers. However, there are no studies investigating the expression and differential roles of WT vs truncated HMGA2 isoforms in prostate cancer. Immunohistochemical staining of prostate tissue microarray revealed low membrane expression in normal epithelial prostate cells, and that expression increased with tumor grade as well as a switch from predominantly cytoplasmic HMGA2 in lower tumor grades, to mostly nuclear in high grade and bone metastatic tissue. LNCaP cells stably overexpressing wild-type HMGA2 displayed nuclear localization of HMGA2 and induction of EMT associated with increased Snail, Twist and vimentin expression compared to LNCaP Neo control cells, as shown by immunofluorescence and western blot analyses. This was associated with increased cell migration on collagen shown using boyden chamber assay. Conversely, LNCaP cells overexpressing truncated HMGA2 showed cytoplasmic HMGA2 expression that did not induce EMT yet displayed increased cell proliferation and migration compared to LNCaP Neo. Both wild-type and truncated HMGA2 increased levels of phospho-ERK, and interestingly, treatment with U0126, MAPK inhibitor, antagonized wild-type HMGA2-mediated EMT and cell migration, but did not affect truncated HMGA2-mediated cell proliferation or migration. Therefore, although both wild-type and truncated HMGA2 may promote prostate tumor progression, wild-type HMGA2 acts by inducing EMT via MAPK pathway.

Triple-Negative Breast Cancers (TNBCs) are the most difficult to treat subtype of breast cancer and are often associated with high nuclear expression of Snail and Cathepsin L (Cat L) protease. We have previously shown that Snail can increase Cat L expression/activity in prostate and breast cancer cells. This study investigated the role of CUX1 (a downstream substrate of Cat L) in TNBC. We showed that Cat L and CUX1 were highly expressed in TNBC patient tissue/cell lines, as compared to ER-positive samples, using cBioportal data and western blot/zymography analyses. Additionally, luciferase reporter and chromatin immunoprecipitation assays showed that CUX1 directly bound to estrogen receptor-alpha (ER-α) promoter in MDA-MB-468, a representative TNBC cell line, and that CUX1 siRNA could restore ER-α transcription and protein expression. Furthermore, Snail and CUX1 expression in various TNBC cell lines was inhibited by muscadine grape skin extract (MSKE, a natural grape product rich in anthocyanins) or Cat L inhibitor (Z-FY-CHO) leading to decreased cell invasion and migration. MSKE decreased cell viability and increased expression of apoptotic markers in MDA-MB-468 cells, with no effect on non-tumorigenic MCF10A cells. MSKE also decreased CUX1 binding to ER-α promoter and restored ER-α expression in TNBC cells, while both MSKE and CUX1 siRNA restored sensitivity to estradiol and 4-hydoxytamoxifen as shown by increased cell viability. Therefore, CUX1 activated by Snail-Cat L signaling may contribute to TNBC via ER-α repression, and may be a viable target for TNBC using natural products such as MSKE that targets cancer and not normal cells.

African Americans (AA) have higher death rates due to prostate and breast cancer as compared to Caucasian Americans (CA), and few biomarkers have been associated with this disparity. In our study we investigated whether epithelial-mesenchymal transition (EMT) with a focus on Snail and Cathepsin L (Cat L), could potentially be two markers associated with prostate and breast health disparities. We have previously shown that Snail can increase Cat L protein and activity in prostate and breast cancer. Western blot and real-time PCR analyses showed that mesenchymal protein expression (Snail, vimentin, Cat L) and Cat L activity (shown by zymography) was higher in AA prostate cancer cells as compared to CA normal transformed RWPE-1 prostate epithelial cells, and androgen-dependent cells, and comparable to metastatic CA cell lines. With respect to breast cancer, mesenchymal markers were higher in TNBC compared to non-TNBC cells. The higher mesenchymal marker expression was functionally associated with higher proliferative and migratory rates. Immunohistochemistry showed that both nuclear Snail and Cat L expression was significantly higher in cancer compared to normal for CA and Bahamas prostate patient tissue. Interestingly, AA normal tissue stained higher for nuclear Snail and Cat L that was not significantly different to cancer tissue for both prostate and breast tissue, but was significantly higher than CA normal tissue. AA TNBC tissue also displayed significantly higher nuclear Snail expression compared to CA TNBC, while no significant differences were observed with Luminal A cancer tissue. Therefore, increased EMT in AA compared to CA that may contribute to the more aggressive disease.

Typically the normal epithelial cells are a single layer, held tightly by adherent proteins that prevent the mobilization of the cells from the monolayer sheet. During prostate cancer progression, the epithelial cells can undergo epithelial-mesenchymal transition or EMT, characterized by morphological changes in their phenotype from cuboidal to spindle-shaped. This is associated with biochemical changes in which epithelial cell markers such as E-cadherin and occludins are down-regulated, which leads to loss of cell-cell adhesion, while mesenchymal markers such as vimentin and N-cadherin are up-regulated, thereby allowing the cells to migrate or metastasize to different organs. The EMT transition can be regulated directly and indirectly by multiple molecular mechanisms including growth factors and cytokines such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF) and insulin-like growth factor (IGF), and signaling pathways such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K). This signaling subsequently induces expression of various transcription factors like Snail, Twist, Zeb1/2, that are also known as master regulators of EMT. Various markers associated with EMT have been reported in prostate cancer patient tissue as well as a possible association with health disparities. There has been consideration to therapeutically target EMT in prostate cancer patients by targeting the EMT signaling pathways.

The purpose of this review is to stimulate new ideas regarding low-dose environmental mixtures and carcinogens and their potential to promote invasion and metastasis. Whereas a number of chapters in this review are devoted to the role of low-dose environmental mixtures and carcinogens in the promotion of invasion and metastasis in specific tumors such as breast and prostate, the overarching theme is the role of low-dose carcinogens in the progression of cancer stem cells. It is becoming clearer that cancer stem cells in a tumor are the ones that assume invasive properties and colonize distant organs. Therefore, low-dose contaminants that trigger epithelial–mesenchymal transition, for example, in these cells are of particular interest in this review. This we hope will lead to the collaboration between scientists who have dedicated their professional life to the study of carcinogens and those whose interests are exclusively in the arena of tissue invasion and metastasis.

Goodson, William H. et al., © The Author 2015. Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology., We gratefully acknowledge the support of the National Institute of Health-National Institute of Environmental Health Sciences (NIEHS) conference grant travel support (R13ES023276); Glenn Rice, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, OH, USA also deserves thanks for his thoughtful feedback and inputs on the manuscript; William H.Goodson III was supported by the California Breast Cancer Research Program, Clarence Heller Foundation and California Pacific Medical Center Foundation; Abdul M.Ali would like to acknowledge the financial support of the University of Sultan Zainal Abidin, Malaysia; Ahmed Lasfar was supported by an award from the Rutgers Cancer Institute of New Jersey; Ann-Karin Olsen and Gunnar Brunborg were supported by the Research Council of Norway (RCN) through its Centres of Excellence funding scheme (223268/F50), Amancio Carnero’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0306-2012); Matilde E. Lleonart was supported by a trienal project grant PI12/01104 and by project CP03/00101 for personal support. Amaya Azqueta would like to thank the Ministerio de Educacion y Ciencia (‘Juande la Cierva’ programme, 2009) of the Spanish Government for personal support; Amedeo Amedei was supported by the Italian Ministry of University and Research (2009FZZ4XM_002), and the University of Florence (ex60%2012); Andrew R.Collins was supported by the University of Oslo; Annamaria Colacci was supported by the Emilia-Romagna Region - Project ‘Supersite’ in Italy; Carolyn Baglole was supported by a salary award from the Fonds de recherche du Quebec-Sante (FRQ-S); Chiara Mondello’s laboratory is supported by Fondazione Cariplo in Milan, Italy (grant n. 2011-0370); Christian C.Naus holds a Canada Research Chair; Clement Yedjou was supported by a grant from the National Institutes of Health (NIH-NIMHD grant no. G12MD007581); Daniel C.Koch is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Award and the Tumor Biology Training grant: NIH T32CA09151; Dean W. Felsher would like to acknowledge the support of United States Department of Health and Human Services, NIH grants (R01 CA170378 PQ22, R01 CA184384, U54 CA149145, U54 CA151459, P50 CA114747 and R21 CA169964); Emilio Rojas would like to thank CONACyT support 152473, Ezio Laconi was supported by AIRC (Italian Association for Cancer Research, grant no. IG 14640) and by the Sardinian Regional Government (RAS); Eun-Yi Moon was supported by grants from the Public Problem-Solving Program (NRF-015M3C8A6A06014500) and Nuclear R&D Program (#2013M2B2A9A03051296 and 2010-0018545) through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (MEST) in Korea; Fahd Al-Mulla was supported by the Kuwait Foundation for the Advancement of Sciences (2011-1302-06); Ferdinando Chiaradonna is supported by SysBioNet, a grant for the Italian Roadmap of European Strategy Forum on Research Infrastructures (ESFRI) and by AIRC (Associazione Italiana Ricerca sul Cancro; IG 15364); Francis L.Martin acknowledges funding from Rosemere Cancer Foundation; he also thanks Lancashire Teaching Hospitals NHS trust and the patients who have facilitated the studies he has undertaken over the course of the last 10 years; Gary S.Goldberg would like to acknowledge the support of the New Jersey Health Foundation; Gloria M.Calaf was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT), Ministerio de Educación de Chile (MINEDUC), Universidad de Tarapacá (UTA); Gudrun Koppen was supported by the Flemish Institute for Technological Research (VITO), Belgium; Hemad Yasaei was supported from a triennial project grant (Strategic Award) from the National Centre for the Replacement, Refinement and Reduction (NC3Rs) of animals in research (NC.K500045.1 and G0800697); Hiroshi Kondoh was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Science and Technology Agency and by JST, CREST; Hsue-Yin Hsu was supported by the Ministry of Science and Technology of Taiwan (NSC93-2314-B-320-006 and NSC94-2314-B-320-002); Hyun Ho Park was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology (2012R1A2A2A01010870) and a grant from the Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (HI13C1449); Igor Koturbash is supported by the UAMS/NIH Clinical and Translational Science Award (UL1TR000039 and KL2TR000063) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; Jan Vondráček acknowledges funding from the Czech Science Foundation (13-07711S); Jesse Roman thanks the NIH for their support (CA116812), John Pierce Wise Sr. and Sandra S.Wise were supported by National Institute of Environmental Health Sciences (ES016893 to J.P.W.) and the Maine Center for Toxicology and Environmental Health; Jonathan Whitfield acknowledges support from the FERO Foundation in Barcelona, Spain; Joseph Christopher is funded by Cancer Research UK and the International Journal of Experimental Pathology; Julia Kravchenko is supported by a philanthropic donation by Fred and Alice Stanback; Jun Sun is supported by a Swim Across America Cancer Research Award; Karine A.Cohen-Solal is supported by a research scholar grant from the American Cancer Society (116683-RSG-09-087-01-TBE); Laetitia Gonzalez received a postdoctoral fellowship from the Fund for Scientific Research–Flanders (FWO-Vlaanderen) and support by an InterUniversity Attraction Pole grant (IAP-P7-07); Laura Soucek is supported by grant #CP10/00656 from the Miguel Servet Research Contract Program and acknowledges support from the FERO Foundation in Barcelona, Spain; Liang-Tzung Lin was supported by funding from the Taipei Medical University (TMU101-AE3-Y19); Linda Gulliver is supported by a Genesis Oncology Trust (NZ) Professional Development Grant, and the Faculty of Medicine, University of Otago, Dunedin, New Zealand; Louis Vermeulen is supported by a Fellowship of the Dutch Cancer Society (KWF, UVA2011-4969) and a grant from the AICR (14–1164); Mahara Valverde would like to thank CONACyT support 153781; Masoud H. Manjili was supported by the office of the Assistant Secretary of Defense for Health Affairs (USA) through the Breast Cancer Research Program under Award No. W81XWH-14-1-0087 Neetu Singh was supported by grant #SR/FT/LS-063/2008 from the Department of Science and Technology, Government of India; Nicole Kleinstreuer is supported by NIEHS contracts (N01-ES 35504 and HHSN27320140003C); P.K. Krishnakumar is supported by the Funding (No. T.K. 11-0629) of King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; Paola A.Marignani is supported by the Dalhousie Medical Research Foundation, The Beatrice Hunter Cancer Institute and CIHR and the Nova Scotia Lung Association; Paul Dent is the holder of the Universal Inc.Chair in Signal Transduction Research and is supported with funds from PHS grants from the NIH (R01-CA141704, R01-CA150214, R01-DK52825 and R01-CA61774); Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, and by the Czech Science Foundation projects P301/12/1686 and 15-03834Y; Po Sing Leung was supported by the Health and Medical Research Fund of Food and Health Bureau, Hong Kong Special Administrative Region, Ref. No: 10110021; Qiang Cheng was supported in part by grant NSF IIS-1218712; R. Brooks Robey is supported by the United States Department of Veterans Affairs; Rabindra Roy was supported by United States Public Health Service Grants (RO1 CA92306, RO1 CA92306-S1 and RO1 CA113447); Rafaela Andrade-Vieira is supported by the Beatrice Hunter Cancer Research Institute and the Nova Scotia Health Research Foundation, Renza Vento was partially funded by European Regional Development Fund, European Territorial Cooperation 2007–13 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–13) and grants from the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2007; Riccardo Di Fiore was a recipient of fellowship granted by European Regional Development Fund, European Territorial Cooperation 2007–2013 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–2013); Rita Dornetshuber-Fleiss was supported by the Austrian Science Fund (FWF, project number T 451-B18) and the Johanna Mahlke, geb.-Obermann-Stiftung; Roberta Palorini is supported by a SysBioNet fellowship; Roslida Abd Hamid is supported by the Ministry of Education, Malaysia-Exploratory Research Grant Scheme-Project no: ERGS/1-2013/5527165; Sabine A.S.Langie is the beneficiary of a postdoctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013; Sakina Eltom is supported by NIH grant SC1CA153326; Samira A.Brooks was supported by National Research Service Award (T32 ES007126) from the National Institute of Environmental Health Sciences and the HHMI Translational Medicine Fellowship; Sandra Ryeom was supported by The Garrett B. Smith Foundation and the TedDriven Foundation; Thierry Massfelder was supported by the Institut National de la Santé et de la Recherche Médicale INSERM and Université de Strasbourg; Thomas Sanderson is supported by the Canadian Institutes of Health Research (CIHR; MOP-115019), the Natural Sciences and Engineering Council of Canada (NSERC; 313313) and the California Breast Cancer Research Program (CBCRP; 17UB-8703); Tiziana Guarnieri is supported by a grant from Fundamental Oriented Research (RFO) to the Alma Mater Studiorum University of Bologna, Bologna, Italy and thanks the Fondazione Cassa di Risparmio di Bologna and the Fondazione Banca del Monte di Bologna e Ravenna for supporting the Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital, Bologna, Italy; W.Kimryn Rathmell is supported by the V Foundation for Cancer Research and the American Cancer Society; William K.Decker was supported in part by grant RP110545 from the Cancer Prevention Research Institute of Texas; William H.Bisson was supported with funding from the NIH P30 ES000210; Yon Rojanasakul was supported with NIH grant R01-ES022968; Zhenbang Chen is supported by NIH grants (MD004038, CA163069 and MD007593); Zhiwei Hu is grateful for the grant support from an institutional start-up fund from The Ohio State University College of Medicine and The OSU James Comprehensive Cancer Center (OSUCCC) and a Seed Award from the OSUCCC Translational Therapeutics Program.

Metastatic prostate cancer (mPCa) relapses after a short period of androgen deprivation therapy and becomes the castration-resistant prostate cancer (CR PCa); to which the treatment is limited. Hence, it is imperative to identify novel therapeutic agents towards this patient population. In the present study, antiproliferative activities of novel imidazopyridines were compared. Among three derivatives, PHE, AMD and AMN, examined, AMD showed the highest inhibitory activity on LNCaP C-81 cell proliferation, following dose- and time-dependent manner. Additionally, AMD exhibited significant antiproliferative effect against a panel of PCa cells, but not normal prostate epithelial cells. Further, when compared to AMD, its derivative DME showed higher inhibitory activities on PCa cell proliferation, clonogenic potential and in vitro tumorigenicity. The inhibitory activity was apparently in part due to the induction of apoptosis. Mechanistic studies indicate that AMD and DME treatments inhibited both AR and PI3K/Akt signaling. The results suggest that better understanding of inhibitory mechanisms of AMD and DME could help design novel therapeutic agents for improving the treatment of CR PCa.

Several recent studies have highlighted an additional unexpected localization and site of action for Cathepsin L (Cat L) protease within the nucleus in breast, colon and prostate cancer, however, its role in the nucleus was unclear. It was proposed to mediate proteolytic processing of the transcription factor CCAAT-displacement protein/cut homeobox transcription factor (Cux1) from the full-length p200 isoform to generate the p110 and p90 isoforms, of which the p110 isoform was shown to act as a cell cycle regulator to accelerate entry into the S phase. The p110 isoform has also been shown to bind to the promoter regions of Snail and E-cadherin to activate Snail and inactivate E-cadherin transcription, thus promoting epithelial mesenchymal transition (EMT). Mechanistic studies on what drives Cat L nuclear localization have not been reported. Our hypothesis is that Snail shuttles into the nucleus with Cat L through binding to importin-β. Snail knockdown with siRNA in MDA-MB-468 breast cancer cells led to nuclear to cytoplasmic shuttling of Cat L and decreased levels of Cux1, while overexpression of Snail in MCF-7 breast cancer cells or HEK-293 human embryonic kidney cells led to increased nuclear expression of both Cat L and Cux1. Additionally, transient transfection of Snail NLS mutants not only abrogated Snail nuclear localization but also nuclear localization of Cat L and Cux1. Interestingly, importin β1 knockdown with siRNA decreased Snail and Cux1 levels, as well as nuclear localization of Cat L. Therefore, we show for the first time that the nuclear localization of Cat L and its substrate Cux1can be positively regulated by Snail NLS and importin β1, suggesting that Snail, Cat L and Cux1 all utilize importin β1 for nuclear import.

Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, possesses antiproliferative and cancer chemopreventive effects. We have demonstrated that the cytostatic/cytotoxic effects of camalexin on several prostate cancer (PCa) cells are due to oxidative stress. Lysosomes are vulnerable organelles to Reactive Oxygen Species (ROS)-induced injuries, with the potential to initiate and or facilitate apoptosis subsequent to release of proteases such as cathepsin D (CD) into the cytosol. We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD. Cell viability was evaluated by MTS cell proliferation assay in LNCaP and ARCaP Epithelial (E) cells, and their respective aggressive sublines C4-2 and ARCaP Mesenchymal (M) cells, whereby the more aggressive PCa cells (C4-2 and ARCaPM) displayed greater sensitivity to camalexin treatments than the lesser aggressive cells (LNCaP and ARCaPE). Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis. Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

High mobility group A (HMGA2), a non-histone protein, is up-regulated in several cancers. Due to chromosomal rearrangement of HMGA2 gene, full-length or wild-type HMGA2 can be truncated leading to loss of the C-terminus and the 3’UTR. Both wild-type and truncated isoforms have been detected in uterine leiomyoma patients. However, the functional role of truncated HMGA2 has not been investigated. We hypothesize that truncated HMGA2 plays a role in prostate cancer progression. We analysed expression of wild-type vs truncated HMGA2 in a panel of prostate cancer cell lines using real-time PCR and western blot analyses. We utilized LNCaP cells stably overexpressing wild-type HMGA2 or truncated HMGA2 in LNCaP cells to detect the basal reactive oxygen species (ROS) levels using DCFDA dye. We analysed Jun-D expression, a putative downstream effector of HMGA2, by western blot analysis, and utilized siRNA to knockdown Jun-D. We performed migration and cell viability assays following Jun-D knockdown. Additionally, we performed proteomic analysis following immunoprecipitation with HMGA2 antibody in nuclear extracts from LNCaP cells overexpressing wild-type HMGA2 WT or cytoplasmic extracts from cells overexpressing truncated HMGA2. Finally, we utilized tissue microarray to analyse expression of HMGA2 by immunohistochemisty. Our results showed that prostate cancer cell lines expressed varying amounts of wild-type and truncated HMGA2. LNCaP cells overexpressing truncated HMGA2 exhibited increased nuclear expression of Jun-D, as well as increased ROS compared to LNCaP cells overexpressing wild-type HMGA2 or empty vector Neo control. Knockdown Jun-D in LNCaP cells overexpressing truncated HMGA2 abrogated ROS induction and cell migration with no effect on cell proliferation. Additionally, truncated HMGA2 interacted with a number of proteins, including Ras GTPase-activating protein-binding protein 1(G3BP1), a cytoplasmic stress granule protein that responds to oxidative stress. Truncated HMGA2 is mainly localized to the cytoplasm, and interestingly we also observed cytoplasmic HMGA expression in prostatic tissue with chronic inflammation. Therefore, truncated HMGA2 may promote prostate cancer progression via Jun-D-mediated ROS. GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22 Citation Format: Ohuod Hawsawi, Veronica Henderson, Liza Burton, Jodi Dougan, Ana Cecillia Millena, Vanessa Adams, Elshaddai Z. White, Guang Di Wang, Qiang Zhang, Cimona Hinton, Shafiq Khan, Valerie Odero-Marah. Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 877.

Prostate Cancer is the most common non-cutaneous cancer and second leading cause of cancer related death in American men. Epithelial Mesenchymal Transition (EMT), a key event in prostate cancer metastasis, is a process in which polarized epithelial cells undergo biochemical changes, allowing them to assume mesenchymal morphologies, enhancing invasiveness and migration. Reactive Oxygen Species (ROS) are chemically reactive molecules of cellular metabolism that induce oxidative stress, and increase levels of SNAI1 (Snail) transcription factor while also promoting EMT. In Phase II clinical trials, patients were treated with natural product, Muscadine Grape Skin Extract (MSKE), and patients with SOD2 Ala/Ala single nucleotide polymorphism (SNP) responded better than those with Val/Val SNP. Ala/Ala SNP is associated with higher dismutase activity [superoxide (O2-) to hydrogen peroxide (H2O2)] than Val/Val. We hypothesize that SOD2 Ala/Ala genotype is associated with increased H2O2 and EMT, potentially antagonized by MSKE in prostate cancer cells. Prostate cancer cell lines were analyzed for SOD2 SNPs by pyrosequencing. Site-directed mutagenesis was conducted to create the Ala/Ala and Val/Val SNP vectors. LNCaP prostate cancer cells were transiently and subsequently stably transfected with SOD2 Val/Val cDNA, as well as empty vector (Neo) control, and verified through western blot analysis. ROS activity was evaluated for baseline ROS activity. Our results showed that most prostate cancer cell lines were heterozygous for SOD2 SNP (Ala/Val), although several had extra copies, for example, LNCaP had Val/Val/Ala, suggesting multiple copy numbers. Only metastatic MDA PCa 2a and MDA PCa 2b contained Ala/Ala homozygous SNP. LNCaP cells transiently and stably transfected with Val/Val showed partial induction of EMT, concomitant with higher levels of Snail and Vimentin, as compared to LNCaP Neo control. Furthermore, data also suggested that transient transfection marginally affected ROS activity while stable transfection caused significant changes in ROS activity compared to LNCaP Neo cells. We have begun to stably transfect Ala/Ala cDNA into LNCaP cells and are interested to see the differences in ROS activity and EMT induction between the two clone cell types (LNCaP Ala/Ala and Val/Val). As previous data suggests, we believe that Ala/Ala will show a higher expression of SOD2 and mesenchymal markers Snail and Vimentin, demonstrating full induction of EMT. In future we will test ROS activity, EMT marker expression in response to treatment of LNCaP Ala/Ala and Val/Val cells with MSKE. These studies may uncover the differential functions and response to treatment of SOD2 SNPs. Acknowledgements: These studies were supported by the NIH/NIMHD/RCI Grant #5G12MD007590-31, NIH/NIGMS/RISE Grant #5R25GM060414, and https://www.thecommunityfoundation.org/ Citation Format: Janae D. Sweeney, Valerie Odero-Marah, Channing Paller. Novel roles for manganese superoxide dismutase polymorphisms in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1047.

Peroxidasin (PXDN), a human homolog of Drosophila PXDN, belongs to the family of heme peroxidases and has been found to promote oxidative stress in cardiovascular tissue, however, its role in prostate cancer has not been previously elucidated. We hypothesized that PXDN promotes prostate cancer progression via regulation of metabolic and oxidative stress pathways. We analyzed PXDN expression in prostate tissue by immunohistochemistry and found increased PXDN expression with prostate cancer progression as compared to normal tissue or cells. PXDN knockdown followed by proteomic analysis revealed an increase in oxidative stress, mitochondrial dysfunction and gluconeogenesis pathways. Additionally, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics confirmed that PXDN knockdown induced global reprogramming associated with increased oxidative stress and decreased nucleotide biosynthesis. We further demonstrated that PXDN knockdown led to an increase in reactive oxygen species (ROS) associated with decreased cell viability and increased apoptosis. Finally, PXDN knockdown decreased colony formation on soft agar. Overall, the data suggest that PXDN promotes progression of prostate cancer by regulating the metabolome, more specifically, by inhibiting oxidative stress leading to decreased apoptosis. Therefore, PXDN may be a biomarker associated with prostate cancer and a potential therapeutic target.

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of vimentin, while epithelial-associated genes like E-cadherin are lost. This enables cells to be more metastatic. Factors that are able to induce EMT include growth factors such as transforming growth factor-β (TGF-β) and epidermal growth factor, and transcription factors such as Snail. Snail-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by the activity of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). LNCaP, 22Rv1 and ARCaP human prostate cancer (CaP) cells stably transfected with empty vector control (Neo) or constitutively active Snail exhibited increased cell invasion. Superarray analysis revealed an upregulation in uPA and uPAR RNA expression in Snail-transfected ARCaP cells compared with that of a Neo control. In addition, the protein expression levels of Snail, uPA and uPAR were measured by western blot analysis which showed that overexpression of Snail increased uPA and uPAR protein levels. The activity of uPA in conditioned media was measured using an ELISA which revealed that uPA activity was elevated in LNCaP, 22Rv1 and ARCaP cells overexpressing Snail. Additionally, transient silencing of uPAR in ARCaP cells overexpressing Snail using short interfering RNA resulted in abrogation of Snail-mediated invasion. Snail overexpression was associated with increased extracellular-signal-regulated kinase activity, and antagonism of this activity with mitogen-activated protein (MAPK) inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human CaP cells may occur via the regulation of uPA/uPAR and the MAPK signaling pathway.

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.

The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression.

Cathepsins are powerful proteases, once referred to as the lysosomal cysteine proteases, that have been implicated in breast cancer invasion and metastasis, but pharmaceutical inhibitors have suffered failures in clinical trials due to adverse side effects. Scientific advancement from lysosomotropic to cell impermeable cathepsin inhibitors have improved efficacy in treating disease, but off-target effects have still been problematic, motivating a need to better understand cellular feedback and responses to treatment with cathepsin inhibitors. To address this need, we investigated effects of E-64 and cystatin C, two broad spectrum cathepsin inhibitors, on cathepsin levels intra- and extracellularly in MDA-MB-231 breast cancer cells. Cathepsins S and L had opposing responses to both E-64 and cystatin C inhibitor treatments with paradoxically elevated amounts of active cathepsin S, but decreased amounts of active cathepsin L, as determined by multiplex cathepsin zymography. This indicated cellular feedback to selectively sustain the amounts of active cathepsin S even in the presence of inhibitors with subnanomolar inhibitory constant values. These differences were identified in cellular locations of cathepsins L and S, trafficking for secretion, co-localization with endocytosed inhibitors, and longer protein turnover time for cathepsin S compared to cathepsin L. Together, this work demonstrates that previously underappreciated cellular compensation and compartmentalization mechanisms may sustain elevated amounts of some active cathepsins while diminishing others after inhibitor treatment. This can confound predictions based solely on inhibitor kinetics, and must be better understood to effectively deploy therapies and dosing strategies that target cathepsins to prevent cancer progression.

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of death from cancer in men. Epithelial-mesenchymal transition (EMT) is a process by which cancer cells invade and migrate, and is characterized by loss of cell-cell adhesion molecules such as E-cadherin and increased expression of mesenchymal proteins such as vimentin; EMT is also associated with resistance to therapy. Snail, a master regulator of EMT, has been extensively studied and reported in cancers such as breast and colon; however, its role in prostate cancer is not as widely reported. The purpose of this review is to put together recent facts that summarize Snail signaling in human prostate cancer. Snail is overexpressed in prostate cancer and its expression and activity is controlled via phosphorylation and growth factor signaling. Snail is involved in its canonical role of inducing EMT in prostate cancer cells; however, it plays a role in non-canonical pathways that do not involve EMT such regulation of bone turnover and neuroendocrine differentiation. Thus, studies indicate that Snail signaling contributes to prostate cancer progression and metastasis and therapeutic targeting of Snail in prostate cancer holds promise in �future.

Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial-mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldeyhyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7 days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3 days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.

Prostate Cancer (PCa) is the second leading cause of cancer death in American men. PCa patients' mortality is mainly attributed to complications caused by metastasis of the disease to organs critical for survival, such as bone. As such, it is important to understand cancer-bone microenvironment interactions in order to develop therapeutics that will slow or halt the process of cancer metastasis. Snail1 is a zinc-finger transcription factor that induces epithelial-mesenchymal transition (EMT) which is associated with cell migration and metastasis in cancer. We hypothesized that cancer cell-bone interactions would promote higher calcium release from bone, more specifically by cancer cells overexpressing Snail, which would lead to increased paracrine cell signaling and migration. For this study, we utilized various prostate cancer cell lines: LNCaP (Snail-low), C4-2 (Snail-high), E006AA (Snail-high), E006AA HT (Snail-high) and C4-2 with stable Snail knockdown. Cancer cells were co-cultured with Hydroxyapatite (HA; inorganic component of bone) of different densities to represent the African American vs Caucasian bone ratio (since African Americans have higher bone density than any other race). The conditioned media was then used to assay calcium levels, perform paracrine migration assays using LNCaP cells, and examine paracrine signaling effects of the various conditioned media on LNCaP cells by western blotting. We observed that calcium levels were elevated in conditioned media from cancer cell-bone co-cultures, compared to media or cancer cells alone, and this could be antagonized by EGTA, a calcium chelator. C4-2 cancer-bone co-culture conditioned media increased paracrine cell migration which was decreased by Snail knockdown as well as lower bone density. We also observed increased STAT3 phosphorylation and paracrine cell migration in LNCaP cells incubated with conditioned media from C4-2, E006AA or E006AA HT cells co-cultured with HA; this phosphorylation and cell migration could be antagonized by Snail knockdown or STAT3 inhibitor (WP1066). An in vivo study was also done using nude mice that were surgically implanted with either 40mg HA or 100mg HA and subcutaneously injected with either C4-2 NS or C4-2 Snail shRNA cells into a position close to the shoulder from the surgical HA implant site in the mouse. At week 2 and week 4, Snail shRNA cells injected mice showed larger tumors than C4-2 NS injected mice, however, mice implanted with cancer cells plus higher bone density resulted in larger tumors than those with lower bone density. In conclusion, our study shows that Snail can mediate cancer-bone microenvironment interactions via STAT3 signaling, that can possibly promote increased paracrine cell migration towards bone of high mineral density. Therefore, targeting cancer-bone micronenvironmental interactions is an important avenue to consider for therapeutic targeting of prostate cancer. Citation Format: Veronica M. Henderson, Ohuod Hawsawi, Liza J. Burton, Kennedi Trice, Jodi Dougan, Simone M. Howard, Valerie A. Odero-Marah. STAT3 pathway regulates the cancer-bone microenvironment interactions mediated by Snail [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1096.

Prostate cancer is the most highly diagnosed cancer in men and the second leading cause of death in the United States. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. High mobility group A (HMGA2), a non-histone protein, has been shown to promote EMT in separate studies. Interestingly, wild-type HMGA2 and truncated (lacking the 3'UTR) HMGA2 isoforms are overexpressed in many cancers, but have not been investigated in prostate cancer. Jun-D is a transcriptional factor that might be regulated by HMGA2. Moreover, the function for each individual isoform is still not well understood. We hypothesize that each of the two isoforms of HMGA2 in will have different functions in prostate cancer. We stably overexpressed wild-type HMGA2 and truncated HMGA2 in LNCaP cells and measured the expression and the localization of EMT markers. We also measured reactive oxygen species (ROS) levels using DCFDA dye, since ROS has been associated with EMT. We performed migration and cell viability assays as well as Jun-D knockdown, a putative downstream effector of HMGA2. Our results showed that the overexpression of HMGA2 in LNCaP cells led to an increase in cell viability and migration for both wild-type and truncated HMGA2. We observed that wild-type, but not truncated HMGA2 promoted EMT. Additionally, Jun-D and ROS levels increased for truncated, but not significantly for wild-type HMGA2. Jun-D knockdown with siRNA decreased ROS and cell migration in cells with truncated HMGA2, but not in cells overexpressing wild-type HMGA2. Therefore, wild-type HMGA2 is the isoform that induces EMT, while the truncated form appears to have an alternate role in upregulating ROS via Jun-D. In conclusion, we show a link between HMGA2 and JunD/ROS for the first time, and data that wild-type and truncated HMGA2 may have differing functions that converge to increase cell proliferation and migration, and may in future instruct on more precise therapeutic targeting in patients with these isoforms. GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22 Citation Format: Ohuod A. Hawsawi, Liza Burton, Jodi Dougan, Ana Cecillia Millena, Peri Nagappan, Shafiq Khan, Valerie Odero-Marah. Role of high mobility group A2 (HMGA2) in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1089.

BACKGROUND Snail transcription factor induces epithelial–mesenchymal transition (EMT) via decreased cell adhesion-associated molecules like E-cadherin, and increased mesenchymal markers like vimentin. We previously established Snail-mediated EMT model utilizing androgen-dependent LNCaP cells. These cells express increased vimentin protein and relocalization of E-cadherin from the cell membrane to the cytosol. Interestingly, Snail transfection in LNCaP cells resulted in cells acquiring a neuroendocrine (NE)-like morphology with long neurite-like processes. METHODS We tested for expression of NE markers neuron-specific enolase (NSE) and chromogranin A (CgA) by Western blot analysis, and performed proliferation assays to test for paracrine cell proliferation. RESULTS LNCaP cells transfected with Snail displayed increase in the NE markers, NSE and CgA as well as translocation of androgen receptor (AR) to the nucleus. LNCaP C-33 cells that have been previously published as a neuroendocrine differentiation (NED) model exhibited increased expression levels of Snail protein as compared to LNCaP parental cells. Functionally, conditioned medium from the LNCaP-Snail transfected cells increased proliferation of parental LNCaP and PC-3 cells, which could be abrogated by NSE/CgA siRNA. Additionally, NED in LNCaP-C33 cells or that induced in parental LNCaP cells by serum starvation could be inhibited by knockdown of Snail with siRNA. CONCLUSION Overall our data provide evidence that Snail transcription factor may promote tumor aggressiveness in the LNCaP cells through multiple processes; induction of EMT may be required to promote migration, while NED may promote tumor proliferation by a paracrine mechanism. Therefore, therapeutic targeting of Snail may prove beneficial in not only abrogating EMT but also NED. Prostate 70: 982–992, 2010. © 2010 Wiley-Liss, Inc.

Zinc-finger enhancer binding protein (ZEB1) is a transcription factor involved in the progression of cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation. We show that ZEB1 and androgen receptor (AR) cross-talk in triple negative breast cancer cell lines. Chromatin immunoprecipitation analysis demonstrates that ZEB1 binds directly to the E-box located in the AR promoter. ZEB1 suppression by stably transfecting shRNA in a triple negative breast cancer cell line resulted in a decrease of AR mRNA, protein, and AR downstream targets. ZEB1 knockdown in triple negative breast cancer cells sensitized the cells to bicalutamide by reducing migration compared to the control cells. Conversely, blockade of AR signaling with bicalutamide resulted in a suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. Furthermore, using a breast cancer tissue microarray, a majority of triple negative breast cancers exhibit positive staining for both ZEB1 and AR. Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell migration in triple negative breast cancer cells.

BACKGROUND Bone morphogenetic proteins (BMPs) exert osteoinductive effects in prostate cancer (PC) via uncharacterized mechanisms. In this study, we investigated whether the nuclear transcription factor NF-κB, implicated in PC metastasis, is involved in transcriptional regulation and activation of BMP-2 or BMP-4/Smad signaling in PC cells. METHODS NF-κB inhibition was achieved by IκBα super-repressor adenoviral vector and activation was monitored by EMSA and reporter assays. BMP expression and activation was measured by PCR and reporter assays. Promoter binding assay was performed by chromatin immunoprecipitation (ChIP) assay. Smad1/5/8 phosphorylation was measured by Western blot analysis. RESULTS PCR and chimeric BMP-2 and BMP-4 luciferase assays demonstrate that NF-κB confers robust and selective activation of BMP-2 in p65 overexpressing or rhTNF-α-stimulated PC cells. Inhibition of NF-κB significantly reduced transcript levels and autocrine production of BMP-2 by rhTNF-α stimulated C4-2B cells and to a lesser extent by the parental LNCaP cells. Selective inhibition of PI3K/Akt suppressed the NF-κB-induced BMP-2 promoter activity. Furthermore, suppression of NF-κB activation decreased the transcript levels and BMP-2-induced phosphorylation of Smad1/5/8, critical downstream targets of BMP-2 signaling in PC cells. Notably, the activation of BMPRII by BMP-2 is required for modulation of Smad activation by NF-κB in PC cells. Based on ChIP analysis, the transcriptional regulation of BMP-2 gene by NF-κB may be partially attributed to binding to κb site on the BMP-2 promoter. CONCLUSIONS The data suggest that PI3K/Akt-NF-κB axis may promote PC bone metastasis in part by regulating transcription and activation of the BMP-2-Smad signaling cascade in osteotropic PC cells. Prostate 69: 168–180, 2009. © 2008 Wiley–Liss, Inc.

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFbeta1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-kappaB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFbeta1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Snail, a zinc-finger transcription factor, induces epithelial-mesenchymal transition (EMT), which is associated with increased cell migration and metastasis in cancer cells. Rac1 is a small G-protein which upon activation results in formation of lamellipodia, the first protrusions formed by migrating cells. We have previously shown that Snail promotes cell migration through down-regulation of maspin tumor suppressor. We hypothesized that Snail's regulation of cell migration may also involve Rac1 signaling regulated by PI3K/AKT and/or MAPK pathways. We found that Snail overexpression in LNCaP and 22Rv1 prostate cancer cells increased Rac1 activity associated with increased cell migration, and the Rac1 inhibitor, NSC23766, could inhibit Snail-mediated cell migration. Conversely, Snail downregulation using shRNA in the aggressive C4-2 prostate cancer cells decreased Rac1 activity and cell migration. Moreover, Snail overexpression increased ERK and PI3K/AKT activity in 22Rv1 prostate cancer cells. Treatment of Snail-overexpressing 22Rv1 cells with LY294002, PI3K/AKT inhibitor or U0126, MEK inhibitor, decreased cell migration significantly, but only LY294002 significantly reduced Rac1 activity, suggesting that Snail promotes Rac1 activation via the PI3K/AKT pathway. Furthermore, 22Rv1 cells overexpressing Snail displayed decreased maspin levels, while inhibition of maspin expression in 22Rv1 cells with siRNA, led to increased PI3K/AKT, Rac1 activity and cell migration, without affecting ERK activity, suggesting that maspin is upstream of PI3K/AKT. Overall, we have dissected signaling pathways by which Snail may promote cell migration through MAPK signaling or alternatively through PI3K/AKT-Rac1 signaling that involves Snail inhibition of maspin tumor suppressor. This may contribute to prostate cancer progression.

Previous studies from our laboratory and others have demonstrated that treatment of breast cancer cells with exogenous maspin led to a significant decrease in cell motility, and an increase in cell adhesion to human fibronectin. However, the signaling mechanisms by which maspin, a putative tumor suppressor gene, might regulate cell motility and adhesion have not been previously addressed. In this study, we hypothesized that maspin could inhibit cell motility through the Rho GTPase pathway, specifically by affecting Rac activity. To test this intriguing hypothesis we utilized an experimental approach where invasive and metastatic MDA-MB-231 breast cancer cells were either treated exogenously with recombinant maspin protein, or stably transfected with maspin. The data revealed decreased Rac1 activity within 4 h, and a decrease in the Rac1 effector, PAK1, within 12 h. In addition, an increase in PI3K and ERK1/2 activities within 1 h of recombinant maspin (rMaspin) treatment was observed, which returned to baseline level after 12 h. ERK activity was shown to be downstream of PI3K, as pretreatment with the PI3K inhibitor, LY294002, inhibited the stimulation of ERK activity by rMaspin. Furthermore, rMaspintreated cells displayed approximately a 30% increase in cell adhesion which was abrogated by pretreatment with LY294002. Increased focal adhesions and stress fibers were observed after 12 h of rMaspin treatment, when the cells were least motile and had reverted to a more epithelial-like phenotype. These data suggest that maspin may inhibit cell motility by regulating Rac1 and subsequently PAK1 activity, and promote cell adhesion via PI3K/ERK pathways. This study provides new insights into the diverse signaling pathways affected by maspin to suppress the metastatic phenotype, and could contribute to novel therapeutic approaches for the treatment of invasive and metastatic breast cancer.