Novel roles for HMGA2 isoforms in regulating oxidative stress and sensitizing to RSL3-Induced ferroptosis in prostate cancer cells.

Oxidative stress is increased in several cancers including prostate cancer, and is currently being exploited in cancer therapy to induce ferroptosis, a novel nonapoptotic form of cell death. High mobility group A2 (HMGA2), a non-histone protein up-regulated in several cancers, can be truncated due to chromosomal rearrangement or alternative splicing of HMGA2 gene. The purpose of this study is to investigate the role of wild-type vs. truncated HMGA2 in prostate cancer (PCa). We analyzed the expression of wild-type vs. truncated HMGA2 and showed that prostate cancer patient tissue and some cell lines expressed increasing amounts of both wild-type and truncated HMGA2 with increasing tumor grade, compared to normal epithelial cells. RNA-Seq analysis of LNCaP prostate cancer cells stably overexpressing wild-type HMGA2 (HMGA2-WT), truncated HMGA2 (HMGA2-TR) or empty vector (Neo) control revealed that HMGA2-TR cells exhibited higher oxidative stress compared to HMGA2-WT or Neo control cells, which was also confirmed by analysis of basal reactive oxygen species (ROS) levels using 2', 7'-dichlorofluorescin diacetate (DCFDA) dye, the ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) and NADP/NADPH using metabolomics. This was associated with increased sensitivity to RAS-selective lethal 3 (RSL3)-induced ferroptosis that could be antagonized by ferrostatin-1. Additionally, proteomic and immunoprecipitation analyses showed that cytoplasmic HMGA2 protein interacted with Ras GTPase-activating protein-binding protein 1 (G3BP1), a cytoplasmic stress granule protein that responds to oxidative stress, and that G3BP1 transient knockdown increased sensitivity to ferroptosis even further. Endogenous knockdown of HMGA2 or G3BP1 in PC3 cells reduced proliferation which was reversed by ferrostatin-1. In conclusion, we show a novel role for HMGA2 in oxidative stress, particularly the truncated HMGA2, which may be a therapeutic target for ferroptosis-mediated prostate cancer therapy.

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth.

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.

Larrea tridentata Extract Mitigates Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

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.

Cancer-bone microenvironmental interactions promotes STAT3 signaling.

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.

Epithelial-Mesenchymal Transition (EMT) and Prostate Cancer.

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-ß), 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.

Association of Epithelial Mesenchymal Transition with prostate and breast health disparities.

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.

High mobility group A2 (HMGA2) promotes EMT via MAPK pathway in prostate cancer.

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.

Snail transcription factor NLS and importin ß1 regulate the subcellular localization of Cathepsin L and Cux1.

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-ß. 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 ß1 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 ß1, suggesting that Snail, Cat L and Cux1 all utilize importin ß1 for nuclear import.

Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor.

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.

Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression.

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.

Snail promotes epithelial mesenchymal transition in breast cancer cells in part via activation of nuclear ERK2.

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Snail mediates invasion through uPA/uPAR and the MAPK signaling pathway in prostate cancer cells.

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-ß (TGF-ß) 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.

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells.

BACKGROUND: Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells. METHODS: Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RESULTS: Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells. CONCLUSIONS: Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

Black and white female adolescents lose vitamin D metabolites into urine.

BACKGROUND: The black American population has a higher prevalence of salt sensitivity compared with the white American population. Dahl salt-sensitive rats, models of salt-induced hypertension, excrete protein-bound vitamin D metabolites into urine, a process that is accelerated during high salt intake. We tested the hypothesis that urinary vitamin D metabolite content and 25-hydroxyvitamin D (25-OHD) binding activity of black female adolescents would be greater than that of white female adolescents. METHODS: Female adolescents (11-15 years old, 11 black and 10 white) were fed low (1.3 g, 56 mmol/24 hours sodium) and high salt (3.86 g, 168 mmol/24 hours sodium) diets for 3 weeks in a randomized order cross-over study design. RESULTS: White and black adolescents had similar mean urinary vitamin D metabolite content (low salt, black versus white: 50 +/- 10 versus 58 +/- 17 pmol/24 hours; high salt, black versus white: 47 +/- 7 versus 79 +/- 16 pmol/24 hours). Mean urinary 25-OHD binding activities of the black and white adolescents did not significantly differ. Urinary 25-OHD binding activity of 10/11 black adolescents and 7/10 white adolescents was greater at week 3 of high salt intake than at week 3 of low salt intake (r = 0.50, P = 0.002, n = 17). Plasma 24,25-dihydroxyvitamin D concentrations of the white female adolescents were significantly higher than that of the black female adolescents (P < 0.001). CONCLUSION: Urinary loss of vitamin D metabolites may be one cause of low vitamin D status, in addition to low dietary intake and reduced skin synthesis.