Chronic Exposure to Low Levels of Parabens Increases Mammary Cancer Growth and Metastasis in Mice.

Methylparaben (MP) and propylparaben (PP) are commonly used as food, cosmetic, and drug preservatives. These parabens are detected in the majority of US women and children, bind and activate estrogen receptors (ER), and stimulate mammary tumor cell growth and invasion in vitro. Hemizygous B6.FVB-Tg (MMTV-PyVT)634Mul/LellJ female mice (n = 20/treatment) were exposed to MP or PP at levels within the US Food and Drug Administration's "human acceptable daily intake." These paraben-exposed mice had increased mammary tumor volume compared with control mice (P < 0.001) and a 28% and 91% increase in the number of pulmonary metastases per week compared with the control mice, respectively (P < 0.0001). MP and PP caused differential expression of 288 and 412 mammary tumor genes, respectively (false discovery rate < 0.05), a subset of which has been associated with human breast cancer metastasis. Molecular docking and luciferase reporter studies affirmed that MP and PP bound and activated human ER, and RNA-sequencing revealed increased ER expression in mammary tumors among paraben-exposed mice. However, ER signaling was not enriched in mammary tumors. Instead, both parabens strongly impaired tumor RNA metabolism (eg, ribosome, spliceosome), as evident from enriched KEGG pathway analysis of differential mammary tumor gene expression common to both paraben treatments (MP, P < 0.001; PP, P < 0.01). Indeed, mammary tumors from PP-exposed mice had an increased retention of introns (P < 0.05). Our data suggest that parabens cause substantial mammary cancer metastasis in mice as a function of their increasing alkyl chain length and highlight the emerging role of aberrant spliceosome activity in breast cancer metastasis.

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners.

Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer's disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aß42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression.

Screening of organoids derived from patients with breast cancer implicates the repressor NCOR2 in cytotoxic stress response and antitumor immunity.

Resistance to antitumor treatment contributes to patient mortality. Functional proteomic screening of organoids derived from chemotherapy-treated patients with breast cancer identified nuclear receptor corepressor 2 (NCOR2) histone deacetylase as an inhibitor of cytotoxic stress response and antitumor immunity. High NCOR2 in the tumors of patients with breast cancer predicted chemotherapy refractoriness, tumor recurrence and poor prognosis. Molecular studies revealed that NCOR2 inhibits antitumor treatment by regulating histone deacetylase 3 (HDAC3) to repress interferon regulatory factor 1 (IRF-1)-dependent gene expression and interferon (IFN) signaling. Reducing NCOR2 or impeding its epigenetic activity by modifying its interaction with HDAC3 enhanced chemotherapy responsiveness and restored antitumor immunity. An adeno-associated viral NCOR2-HDAC3 competitor potentiated chemotherapy and immune checkpoint therapy in culture and in vivo by permitting transcription of IRF-1-regulated proapoptosis and inflammatory genes to increase IFN-γ signaling. The findings illustrate the utility of patient-derived organoids for drug discovery and suggest that targeting stress and inflammatory-repressor complexes such as NCOR2-HDAC3 could overcome treatment resistance and improve the outcome of patients with cancer.

WWOX and Its Binding Proteins in Neurodegeneration.

WW domain-containing oxidoreductase (WWOX) is known as one of the risk factors for Alzheimer's disease (AD), a neurodegenerative disease. WWOX binds Tau via its C-terminal SDR domain and interacts with Tau phosphorylating enzymes ERK, JNK, and GSK-3ß, and thereby limits AD progression. Loss of WWOX in newborns leads to severe neural diseases and early death. Gradual loss of WWOX protein in the hippocampus and cortex starting from middle age may slowly induce aggregation of a protein cascade that ultimately causes accumulation of extracellular amyloid beta plaques and intracellular tau tangles, along with reduction in inhibitory GABAergic interneurons, in AD patients over 70 years old. Age-related increases in pS14-WWOX accumulation in the brain promotes neuronal degeneration. Suppression of Ser14 phosphorylation by a small peptide Zfra leads to enhanced protein degradation, reduction in NF-κB-mediated inflammation, and restoration of memory loss in triple transgenic mice for AD. Intriguingly, tumor suppressors p53 and WWOX may counteract each other in vivo, which leads to upregulation of AD-related protein aggregation in the brain and lung. WWOX has numerous binding proteins. We reported that the stronger the binding between WWOX and its partners, the better the suppression of cancer growth and reduction in inflammation. In this regard, the stronger complex formation between WWOX and partners may provide a better blockade of AD progression. In this review, we describe whether and how WWOX and partner proteins control inflammatory response and protein aggregation and thereby limit AD progression.

Corrigendum: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

[This corrects the article DOI: 10.3389/fonc.2021.638311.].

A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

INTRODUCTION: Stem-like cancer cells or cancer stem cells (CSCs) may comprise a phenotypically and functionally heterogeneous subset of cells, whereas the molecular markers reflecting this CSC hierarchy remain elusive. The glycolytic enzyme alpha-enolase (ENO1) present on the surface of malignant tumor cells has been identified as a metastasis-promoting factor through its function of activating plasminogen. The expression pattern of surface ENO1 (sENO1) concerning cell-to-cell or CSC heterogeneity and its functional roles await further investigation. METHODS: The cell-to-cell expression heterogeneity of sENO1 was profiled in malignant cells from different types of cancers using flow cytometry. The subcellular localization of sENO1 and its functional roles in the invadopodia formation and cancer cell invasiveness were investigated using a series of imaging, molecular, and in vitro and in vivo functional studies. RESULTS: We showed here that ENO1 is specifically localized to the invadopodial surface of a significant subset (11.1%-63.9%) of CSCs in human gastric and prostate adenocarcinomas. sENO1+ CSCs have stronger mesenchymal properties than their sENO1- counterparts. The subsequent functional studies confirmed the remarkable pro-invasive and pro-metastatic capacities of sENO1+ CSCs. Mechanistically, inhibiting the surface localization of ENO1 by downregulating caveolin-1 expression compromised invadopodia biogenesis, proteolysis, and CSC invasiveness. CONCLUSIONS: Our study identified the specific expression of ENO1 on the invadopodial surface of a subset of highly invasive and pro-metastatic CSCs. sENO1 may provide a diagnostically and/or therapeutically exploitable target to improve the outcome of patients with aggressive and metastatic cancers.

Therapeutic Zfra4-10 or WWOX7-21 Peptide Induces Complex Formation of WWOX with Selective Protein Targets in Organs that Leads to Cancer Suppression and Spleen Cytotoxic Memory Z Cell Activation In Vivo.

Synthetic Zfra4-10 and WWOX7-21 peptides strongly suppress cancer growth in vivo. Hypothetically, Zfra4-10 binds to the membrane Hyal-2 of spleen Z cells and activates the Hyal-2/WWOX/SMAD4 signaling for cytotoxic Z cell activation to kill cancer cells. Stimulation of membrane WWOX in the signaling complex by a WWOX epitope peptide, WWOX7-21, is likely to activate the signaling. Here, mice receiving Zfra4-10 or WWOX7-21 peptide alone exhibited an increased binding of endogenous tumor suppressor WWOX with ERK, C1qBP, NF-κB, Iba1, p21, CD133, JNK1, COX2, Oct4, and GFAP in the spleen, brain, and/or lung which led to cancer suppression. However, when in combination, Zfra4-10 and WWOX7-21 reduced the binding of WWOX with target proteins and allowed tumor growth in vivo. In addition to Zfra4-10 and WWOX7-21 peptides, stimulating the membrane Hyal-2/WWOX complex with Hyal-2 antibody and sonicated hyaluronan (HAson) induced Z cell activation for killing cancer cells in vivo and in vitro. Mechanistically, Zfra4-10 binds to membrane Hyal-2, induces dephosphorylation of WWOX at pY33 and pY61, and drives Z cell activation for the anticancer response. Thus, Zfra4-10 and WWOX7-21 peptides, HAson, and the Hyal-2 antibody are of therapeutic potential for cancer suppression.

Functional role of WW domain-containing proteins in tumor biology and diseases: Insight into the role in ubiquitin-proteasome system.

The ubiquitin-proteasome system (UPS) governs the protein degradation process and balances proteostasis and cellular homeostasis. It is a well-controlled mechanism, in which removal of the damaged or excessive proteins is essential in driving signal pathways for cell survival or death. Accumulation of damaged proteins and failure in removal may contribute to disease initiation such as in cancers and neurodegenerative diseases. In this notion, specific protein-protein interaction is essential for the recognition of targeted proteins in UPS. WW domain plays an indispensable role in the protein-protein interactions during signaling. Among the 51 WW domain-containing proteins in the human proteomics, near one-quarter of them are involved in the UPS, suggesting that WW domains are crucial modules for driving the protein-protein binding and subsequent ubiquitination and degradation. In this review, we detail a broad spectrum of WW domains in protein-protein recognition, signal transduction, and relevance to diseases. New perspectives in dissecting the molecular interactions are provided.

Chasing the signaling run by tri-molecular time-lapse FRET microscopy.

A feasible design is made to measure three protein/protein interactions to visualize signal pathways by time-lapse Förster resonance energy transfer (FRET) microscopy. When interacting proteins are in close proximity, excitation energy is provided to allow the energy flow from the first molecule to excite the second, followed by energy transfer to the third. By phorbol ester/calcium ionophore stimulation, for example, a real-time complex formation of ectopic IκBα/ERK/WWOX occurs as measured by FRET microscopy, indicative of an ongoing functional signaling. Hyaluronan induces membrane Hyal-2 signaling, which allows FRET measurement of the complex formation of ectopic Smad4/WWOX/Hyal-2 for causing bubbling cell death. If ectopic p53 is recruited to replace Hyal-2, the resulting ectopic Smad4/WWOX/p53 complex induces membrane blebbing without cell death. Together, in this perspective review article, we demonstrate the utilization of time-lapse FRET microscopy to visualize the signaling event via the tri-molecular protein complex formation and their biological outcomes. We show an initial two-protein binding to form the driving force to jumpstart the tri-molecular execution for the signal pathway.

Phosphorylation/de-phosphorylation in specific sites of tumor suppressor WWOX and control of distinct biological events.

Abnormal differentiation and growth of hematopoietic stem cells cause the development of hematopoietic diseases and hematopoietic malignancies. However, the molecular events underlying leukemia development are not well understood. In our recent study, we have demonstrated that calcium ionophore and phorbol ester force the differentiation of T lymphoblastic leukemia. The event involves a newly identified IκBα/WWOX/ERK signaling, in which WWOX is Ser14 phosphorylated. Additional evidence also reveals that pS14-WWOX is involved in enhancing cancer progression and metastasis and facilitating neurodegeneration. In this mini-review, we update the current knowledge for the functional roles of WWOX under physiological and pathological settings, and provide new insights regarding pS14-WWOX in T leukemia cell maturation, and switching the anticancer pY33-WWOX to pS14-WWOX for cancer promotion and disease progression. Impact statement WWOX was originally designated as a tumor suppressor. However, human newborns deficient in WWOX do not spontaneously develop tumors. Activated WWOX with Tyr33 phosphorylation is present in normal tissues and organs. However, when pY33-WWOX is overly induced under stress conditions, it becomes apoptotic to eliminate damaged cells. Notably, WWOX with Ser14 phosphorylation is upregulated in the lesions of cancer, as well as in the brain hippocampus and cortex with Alzheimer's disease. Suppression of pS14-WWOX by Zfra reduces cancer growth and mitigates Alzheimer's disease progression, suggesting that pS14-WWOX facilitates disease progression. pS14-WWOX can be regarded as a marker of disease progression.

Metronomic chemotherapy prevents therapy-induced stromal activation and induction of tumor-initiating cells.

Although traditional chemotherapy kills a fraction of tumor cells, it also activates the stroma and can promote the growth and survival of residual cancer cells to foster tumor recurrence and metastasis. Accordingly, overcoming the host response induced by chemotherapy could substantially improve therapeutic outcome and patient survival. In this study, resistance to treatment and metastasis has been attributed to expansion of stem-like tumor-initiating cells (TICs). Molecular analysis of the tumor stroma in neoadjuvant chemotherapy-treated human desmoplastic cancers and orthotopic tumor xenografts revealed that traditional maximum-tolerated dose chemotherapy, regardless of the agents used, induces persistent STAT-1 and NF-κB activity in carcinoma-associated fibroblasts. This induction results in the expression and secretion of ELR motif-positive (ELR+) chemokines, which signal through CXCR-2 on carcinoma cells to trigger their phenotypic conversion into TICs and promote their invasive behaviors, leading to paradoxical tumor aggression after therapy. In contrast, the same overall dose administered as a low-dose metronomic chemotherapy regimen largely prevented therapy-induced stromal ELR+ chemokine paracrine signaling, thus enhancing treatment response and extending survival of mice carrying desmoplastic cancers. These experiments illustrate the importance of stroma in cancer therapy and how its impact on treatment resistance could be tempered by altering the dosing schedule of systemic chemotherapy.

Role of WW Domain-containing Oxidoreductase WWOX in Driving T Cell Acute Lymphoblastic Leukemia Maturation.

Whether tumor suppressor WWOX (WW domain-containing oxidoreductase) stimulates immune cell maturation is largely unknown. Here, we determined that Tyr-33-phosphorylated WWOX physically binds non-phosphorylated ERK and IκBα in immature acute lymphoblastic leukemia MOLT-4 T cells and in the naïve mouse spleen. The IκBα·ERK·WWOX complex was shown to localize, in part, in the mitochondria. WWOX prevents IκBα from proteasomal degradation. Upon stimulating MOLT-4 with ionophore A23187/phorbol myristate acetate, endogenous IκBα and ERK undergo rapid phosphorylation in <5 min, and subsequently WWOX is Tyr-33 and Tyr-287 de-phosphorylated and Ser-14 phosphorylated. Three hours later, IκBα starts to degrade, and ERK returns to basal or non-phosphorylation, and this lasts for the next 12 h. Finally, expression of CD3 and CD8 occurs in MOLT-4 along with reappearance of the IκBα·ERK·WWOX complex near 24 h. Inhibition of ERK phosphorylation by U0126 or IκBα degradation by MG132 prevents MOLT-4 maturation. By time-lapse FRET microscopy, IκBα·ERK·WWOX complex exhibits an increased binding strength by 1-2-fold after exposure to ionophore A23187/phorbol myristate acetate for 15-24 h. Meanwhile, a portion of ERK and WWOX relocates to the nucleus, suggesting their role in the induction of CD3 and CD8 expression in MOLT-4.

UV irradiation/cold shock-mediated apoptosis is switched to bubbling cell death at low temperatures.

When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

Zfra activates memory Hyal-2+ CD3- CD19- spleen cells to block cancer growth, stemness, and metastasis in vivo.

Zfra is a 31-amino-acid zinc finger-like protein, which participates in the tumor necrosis factor signaling. Here, we determined that when nude mice and BALB/c mice were pre-injected with nanogram levels of a synthetic Zfra1-31 or truncated Zfra4-10 peptide via tail veins, these mice became resistant to the growth, metastasis and stemness of melanoma cells, and many malignant cancer cells. The synthetic peptides underwent self-polymerization in phosphate-buffered saline. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra aggregation and its-mediated cancer suppression in vivo. Injected Zfra peptide autofluoresced due to polymerization and was trapped mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naïve mice conferred resistance to cancer growth. Zfra-binding cells, designated Hyal-2+ CD3- CD19- Z cells, are approximately 25-30% in the normal spleen, but are significantly downregulated (near 0-3%) in tumor-growing mice. Zfra prevented the loss of Z cells caused by tumors. In vitro stimulation or education of naïve spleen cells with Zfra allowed generation of activated Z cells to confer a memory anticancer response in naïve or cancer-growing mice. In particular, Z cells are abundant in nude and NOD-SCID mice, and can be readily activated by Zfra to mount against cancer growth.

A gene expression signature of epithelial tubulogenesis and a role for ASPM in pancreatic tumor progression.

BACKGROUND & AIMS: Many patients with pancreatic ductal adenocarcinoma (PDAC) develop recurrent or metastatic diseases after surgery, so it is important to identify those most likely to benefit from aggressive therapy. Disruption of tissue microarchitecture is an early step in pancreatic tumorigenesis and a parameter used in pathology grading of glandular tumors. We investigated whether changes in gene expression during pancreatic epithelial morphogenesis were associated with outcomes of patients with PDAC after surgery. METHODS: We generated architectures of human pancreatic duct epithelial cells in a 3-dimensional basement membrane matrix. We identified gene expression profiles of the cells during different stages of tubular morphogenesis (tubulogenesis) and of PANC-1 cells during spheroid formation. Differential expression of genes was confirmed by immunoblot analysis. We compared the gene expression profile associated with pancreatic epithelial tubulogenesis with that of PDAC samples from 27 patients, as well as with their outcomes after surgery. RESULTS: We identified a gene expression profile associated with tubulogenesis that resembled the profile of human pancreatic tissue with differentiated morphology and exocrine function. Patients with PDACs with this profile fared well after surgery. Based on this profile, we established a 6-28 gene tubulogenesis-specific signature that accurately determined the prognosis of independent cohorts of patients with PDAC (total n = 128; accuracy = 81.2%-95.0%). One gene, ASPM, was down-regulated during tubulogenesis but up-regulated in human PDAC cell lines and tumor samples; up-regulation correlated with patient outcomes (Cox regression P = .0028). Bioinformatic, genetic, biochemical, functional, and clinical correlative studies showed that ASPM promotes aggressiveness of PDAC by maintaining Wnt-ß-catenin signaling and stem cell features of PDAC cells. CONCLUSIONS: We identified a gene expression profile associated with pancreatic epithelial tubulogenesis and a tissue architecture-specific signature of PDAC cells that is associated with patient outcomes after surgery.

Role of WWOX and NF-κB in lung cancer progression.

It is generally agreed that the pro-inflammatory, pro-survival transcription factor NF-κB is a tumor promoter. Tumor necrosis factor alpha (TNF-α or TNF) mediates NF-κB activation. Tumor suppressor WWOX (FOR or WOX1) is a downstream effector of the TNF signaling. Thus, activation of both WWOX (FOR or WOX1) and NF-κB may occur during TNF signaling and/or under stress conditions. Indeed, the first WW domain of WWOX induces the activation of NF-κB-responsive promoter without TNF participation. It appears that WWOX counteracts with NF-κB in regulating cell survival and death. For example, WWOX becomes activated with Tyr33 phosphorylation and relocates together with NF-κB and many transcription factors to the nucleus to cause neuronal death in sciatic nerve-transected rats. While WWOX is frequently lost in lung cancer and many other cancers, NF-κB activation-induced cancer promotion probably requires WWOX-independent signaling networks to induce expression of pro-survival factors. The antagonistic role of WWOX and NF-κB in the regulation of lung cancer progression is discussed.

Identification of an In Vivo MEK/WOX1 Complex as a Master Switch for Apoptosis in T Cell Leukemia.

Not all leukemia T cells are susceptible to high levels of phorbol myristate acetate (PMA)-mediated apoptosis. At micromolar levels, PMA induces apoptosis of Jurkat T cells by causing mitochondrial polarization/de-polarization, release of cytosolic granules, and DNA fragmentation. Chemical inhibitors U0126 and PD98059 block mitogen-activated protein kinase kinase 1 (MEK1)-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and prevent apoptosis. Mechanistically, proapoptotic tumor suppressor WOX1 (also named WWOX or FOR) physically interacts with MEK1, in part, in the lysosomes in Jurkat cells. PMA induces the dissociation, which leads to relocation of MEK1 to lipid rafts and WOX1 to the mitochondria for causing apoptosis. U0126 inhibits PMA-induced dissociation of WOX1/MEK1 complex and supports survival of Jurkat cells. In contrast, less differentiated Molt-4 T cells are resistant to PMA-induced dissociation of the WOX1/MEK1 complex and thereby are refractory to apoptosis. U0126 overturns the resistance for enhancing apoptosis in Molt-4 cells. Together, the in vivo MEK1/WOX1 complex is a master on/off switch for apoptosis in leukemia T cells.