Hypoxia-activated XBP1s recruits HDAC2-EZH2 to engage epigenetic suppression of ΔNp63α expression and promote breast cancer metastasis independent of HIF1α.

Hypoxia is a hallmark of cancer development. However, the molecular mechanisms by which hypoxia promotes tumor metastasis are not fully understood. In this study, we demonstrate that hypoxia promotes breast cancer metastasis through suppression of ΔNp63α in a HIF1α-independent manner. We show that hypoxia-activated XBP1s forms a stable repressor protein complex with HDAC2 and EZH2 to suppress ΔNp63α transcription. Notably, H3K27ac is predominantly occupied on the ΔNp63 promoter under normoxia, while H3K27me3 on the promoter under hypoxia. We show that XBP1s binds to the ΔNp63 promoter to recruit HDAC2 and EZH2 in facilitating the switch of H3K27ac to H3K27me3. Pharmacological inhibition or the knockdown of either HDAC2 or EZH2 leads to increased H3K27ac, accompanied by the reduced H3K27me3 and restoration of ΔNp63α expression suppressed by hypoxia, resulting in inhibition of cell migration. Furthermore, the pharmacological inhibition of IRE1α, but not HIF1α, upregulates ΔNp63α expression in vitro and inhibits tumor metastasis in vivo. Clinical analyses reveal that reduced p63 expression is correlated with the elevated expression of XBP1, HDAC2, or EZH2, and is associated with poor overall survival in human breast cancer patients. Together, these results indicate that hypoxia-activated XBP1s modulates the epigenetic program in suppression of ΔNp63α to promote breast cancer metastasis independent of HIF1α and provides a molecular basis for targeting the XBP1s/HDAC2/EZH2-ΔNp63α axis as a putative strategy in the treatment of breast cancer metastasis.

FBXO3 stabilizes USP4 and Twist1 to promote PI3K-mediated breast cancer metastasis.

Tumor metastasis is the major cause of breast cancer morbidity and mortality. It has been reported that the F-box protein FBXO3 functions as an E3 ubiquitin ligase in regulating various biological processes, including host autoimmune, antiviral innate immunity, and inflammatory response. However, the role of FBXO3 in tumor metastasis remains elusive. We have previously shown that ΔNp63α is a common inhibitory target in oncogene-induced cell motility and tumor metastasis. In this study, we show that FBXO3 plays a vital role in PI3K-mediated breast cancer metastasis independent of its E3 ligase activity and ΔNp63α in breast cancer cells and in mouse. FBXO3 can bind to and stabilize USP4, leading to Twist1 protein stabilization and increased breast cancer cell migration and tumor metastasis. Mechanistically, FBXO3 disrupts the interaction between USP4 and aspartyl aminopeptidase (DNPEP), thereby protecting USP4 from DNPEP-mediated degradation. Furthermore, p110αH1047R facilitates the phosphorylation and stabilization of FBXO3 in an ERK1-dependent manner. Knockdown of either FBXO3 or USP4 leads to significant inhibition of PI3K-induced breast cancer metastasis. Clinically, elevated expression of p110α/FBXO3/USP4/Twist1 is associated with poor overall survival (OS) and recurrence-free survival (RFS) of breast cancer patients. Taken together, this study reveals that the FBXO3-USP4-Twist1 axis is pivotal in PI3K-mediated breast tumor metastasis and that FBXO3/USP4 may be potential therapeutic targets for breast cancer treatment.

USP36 stabilizes nucleolar Snail1 to promote ribosome biogenesis and cancer cell survival upon ribotoxic stress.

Tumor growth requires elevated ribosome biogenesis. Targeting ribosomes is an important strategy for cancer therapy. The ribosome inhibitor, homoharringtonine (HHT), is used for the clinical treatment of leukemia, yet it is ineffective for the treatment of solid tumors, the reasons for which remain unclear. Here we show that Snail1, a key factor in the regulation of epithelial-to-mesenchymal transition, plays a pivotal role in cellular surveillance response upon ribotoxic stress. Mechanistically, ribotoxic stress activates the JNK-USP36 signaling to stabilize Snail1 in the nucleolus, which facilitates ribosome biogenesis and tumor cell survival. Furthermore, we show that HHT activates the JNK-USP36-Snail1 axis in solid tumor cells, but not in leukemia cells, resulting in solid tumor cell resistance to HHT. Importantly, a combination of HHT with the inhibition of the JNK-USP36-Snail1 axis synergistically inhibits solid tumor growth. Together, this study provides a rationale for targeting the JNK-USP36-Snail1 axis in ribosome inhibition-based solid tumor therapy.

Intratumoral Microbiota Composition Regulates Chemoimmunotherapy Response in Esophageal Squamous Cell Carcinoma.

Neoadjuvant chemoimmunotherapy (NACI) has shown promise in the treatment of resectable esophageal squamous cell carcinoma (ESCC). The microbiomes of patients can impact therapy response, and previous studies have demonstrated that intestinal microbiota influences cancer immunotherapy by activating gut immunity. Here, we investigated the effects of intratumoral microbiota on the response of patients with ESCC to NACI. Intratumoral microbiota signatures of ß-diversity were disparate and predicted the treatment efficiency of NACI. The enrichment of Streptococcus positively correlated with GrzB+ and CD8+ T-cell infiltration in tumor tissues. The abundance of Streptococcus could predict prolonged disease-free survival in ESCC. Single-cell RNA sequencing demonstrated that responders displayed a higher proportion of CD8+ effector memory T cells but a lower proportion of CD4+ regulatory T cells. Mice that underwent fecal microbial transplantation or intestinal colonization with Streptococcus from responders showed enrichment of Streptococcus in tumor tissues, elevated tumor-infiltrating CD8+ T cells, and a favorable response to anti-PD-1 treatment. Collectively, this study suggests that intratumoral Streptococcus signatures could predict NACI response and sheds light on the potential clinical utility of intratumoral microbiota for cancer immunotherapy. SIGNIFICANCE: Analysis of intratumoral microbiota in patients with esophageal cancer identifies a microbiota signature that is associated with chemoimmunotherapy response and reveals that Streptococcus induces a favorable response by stimulating CD8+ T-cell infiltration. See related commentary by Sfanos, p. 2985.

ER stress activates TAp73α to promote colon cancer cell apoptosis via the PERK-ATF4 pathway.

Colorectal cancer (CRC) is the fourth most diagnosed cancer worldwide. 43% of CRCs harbor p53 mutations. The tumor suppressor p53 induces cell growth arrest and/or apoptosis in response to stress, including endoplasmic reticulum (ER) stress. It has been documented that the p53 gene is mutated in more than 50% of human tumors and loses its tumor suppressor function, suggesting that ER stress-induced apoptosis might not rely on p53. In this study, we found that activation of ER stress promotes p53 null colon cancer cell apoptosis concomitant with an increased level of the TAp73α protein, a homologue of p53 in vitro and in vivo. Knockdown of TAp73α partially restores ER stress-induced apoptosis, indicating that ER stress stimulates apoptosis in a manner dependent on TAp73α, but not p53. Furthermore, we found that ER stress activates TAp73α mRNA and protein expression through PERK signalling, a branch of the unfolded protein response (UPR). Moreover, PERK promotes TAp73α expression by upregulating the expression of the transcription factor ATF4. ATF4 directly activates the transcription of TAp73α. Consistent with this finding, ATF4 knockdown inhibited PERK- or ER stress-induced TAp73α expression. Our findings reveal that ER stress activates TAp73α to promote colon cancer cell apoptosis via the PERK-ATF4 signalling. Therefore, prolonged ER stress or upregulation of TAp73α might be a therapeutic strategy for colon cancer.

HSF1 activates the FOXO3a-ΔNp63α-CDK4 axis to promote head and neck squamous cell carcinoma cell proliferation and tumour growth.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers worldwide. Heat shock factor 1 (HSF1) is a conserved transcriptional factor that plays a critical role in maintaining cellular proteostasis. However, the role of HSF1 in HNSCC development remains largely unclear. Here, we report that HSF1 promotes forkhead box protein O3a (FOXO3a)-dependent transcription of ΔNp63α (p63 isoform in the p53 family; inhibits cell migration, invasion, and metastasis), which leads to upregulation of cyclin-dependent kinase 4 expression and HNSCC tumour growth. Ablation of HSF1 or treatment with KRIBB11, a specific pharmacological inhibitor of HSF1, significantly suppresses ΔNp63α expression and HNSCC tumour growth. Clinically, the expression of HSF1 is positively correlated with the expression of ΔNp63α in HNSCC tumours. Together, this study demonstrates that the HSF1-ΔNp63α pathway is critically important for HNSCC tumour growth.

FBXL2 promotes E47 protein instability to inhibit breast cancer stemness and paclitaxel resistance.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of metastasis and recurrence. Although chemotherapy has greatly improved the clinical outcome of TNBC patients, acquired drug resistance remains a huge challenge for TNBC treatment. Breast cancer stem cells (BCSCs) play a critical role in breast cancer development, metastasis, recurrence, and chemotherapy resistance. Thus, it is of great importance to decipher the underlying molecular mechanism of BCSCs regulation for TNBC drug resistance. In this study, we demonstrate that the F-box protein FBXL2 is a critical negative regulator of BCSCs stemness and that downregulation of FBXL2 plays a causal role in TNBC drug resistance. We show that expression levels of FBXL2 significantly influence CD44high/CD24low subpopulation and the mammosphere formation ability of TNBC cells. Ectopic expression of FBXL2 inhibits initiation of TNBC and overcomes paclitaxel resistance in vivo. In addition, activation of FBXL2 by nebivolol, a clinically used small-molecule inhibitor of the beta-1 receptor, markedly overcomes BCSCs-induced paclitaxel resistance. Mechanistically, we show that FBXL2 targets transcriptional factor E47 for polyubiquitin- and proteasome-mediated degradation, resulting in inhibition of BCSC stemness. Clinical analyses indicate that low expression of FBXL2 correlates with high expression of E47 as well as with high stemness features, and is associated with poor clinical outcomes of breast cancer patients. Taken together, these results highlight that the FBXL2-E47 axis plays a critical role in the regulation of BCSC stemness and paclitaxel resistance. Thus, targeting FBXL2 might be a potential therapeutic strategy for drug-resistant TNBC.

USP5-Beclin 1 axis overrides p53-dependent senescence and drives Kras-induced tumorigenicity.

Non-small cell lung cancers (NSCLC) frequently contain KRAS mutation but retain wild-type TP53. Abundant senescent cells are observed in premalignant but not in malignant tumors derived from the Kras-driven mouse model, suggesting that KRAS oncogenic signaling would have to overcome the intrinsic senescence burden for cancer progression. Here, we show that the nuclear Beclin 1-mediated inhibition of p53-dependent senescence drives Kras-mediated tumorigenesis. KRAS activates USP5 to stabilize nuclear Beclin 1, leading to MDM2-mediated p53 protein instability. KrasG12D mice lacking Beclin 1 display retarded lung tumor growth. Knockdown of USP5 or knockout of Becn1 leads to increased senescence and reduced autophagy. Mechanistically, KRAS elevates ROS to induce USP5 homodimer formation by forming the C195 disulfide bond, resulting in stabilization and activation of USP5. Together, these results demonstrate that activation of the USP5-Beclin 1 axis is pivotal in overriding intrinsic p53-dependent senescence in Kras-driven lung cancer development.

Correction: Gao et al. TGF-ß1 Facilitates TAp63α Protein Lysosomal Degradation to Promote Pancreatic Cancer Cell Migration. Biology 2021, 10, 597.

In the original publication [...].

Nuclear Beclin 1 Destabilizes Retinoblastoma Protein to Promote Cell Cycle Progression and Colorectal Cancer Growth.

Autophagy is elevated in colorectal cancer (CRC) and is generally associated with poor prognosis. However, the role of autophagy core-protein Beclin 1 remains controversial in CRC development. Here, we show that the expression of nuclear Beclin 1 protein is upregulated in CRC with a negative correlation to retinoblastoma (RB) protein expression. Silencing of BECN1 upregulates RB resulting in cell cycle G1 arrest and growth inhibition of CRC cells independent of p53. Furthermore, ablation of BECN1 inhibits xenograft tumor growth through elevated RB expression and reduced autophagy, while simultaneous silencing of RB1 restores tumor growth but has little effect on autophagy. Mechanistically, knockdown of BECN1 promotes the complex formation of MDM2 and MDMX, resulting in MDM2-dependent MDMX instability and RB stabilization. Our results demonstrate that nuclear Beclin 1 can promote cell cycle progression through modulation of the MDM2/X-RB pathway and suggest that Beclin 1 promotes CRC development by facilitating both cell cycle progression and autophagy.

The Hsp70-Bag3 complex modulates the phosphorylation and nuclear translocation of Hippo pathway protein Yap.

Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70-Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.

FBXL2 counteracts Grp94 to destabilize EGFR and inhibit EGFR-driven NSCLC growth.

Abnormal activation of epidermal growth factor receptor (EGFR) drives non-small cell lung cancer (NSCLC) development. EGFR mutations-mediated resistance to tyrosine-kinase inhibitors (TKIs) is a major hurdle for NSCLC treatment. Here, we show that F-box protein FBXL2 targets EGFR and EGFR TKI-resistant mutants for proteasome-mediated degradation, resulting in suppression of EGFR-driven NSCLC growth. Reduced FBXL2 expression is associated with poor clinical outcomes of NSCLC patients. Furthermore, we show that glucose-regulated protein 94 (Grp94) protects EGFR from degradation via blockage of FBXL2 binding to EGFR. Moreover, we have identified nebivolol, a clinically used small molecule inhibitor, that can upregulate FBXL2 expression to inhibit EGFR-driven NSCLC growth. Nebivolol in combination with osimertinib or Grp94-inhibitor-1 exhibits strong inhibitory effects on osimertinib-resistant NSCLC. Together, this study demonstrates that the FBXL2-Grp94-EGFR axis plays a critical role in NSCLC development and suggests that targeting FBXL2-Grp94 to destabilize EGFR may represent a putative therapeutic strategy for TKI-resistant NSCLC.

TGF-ß1 Facilitates TAp63α Protein Lysosomal Degradation to Promote Pancreatic Cancer Cell Migration.

TGF-ß signaling plays a pivotal role in promoting tumor cell migration and cancer metastasis. ΔNp63α and TAp63α are two major isoforms of p53-related p63 protein. Our recent study has shown that TGF-ß1 promotes ΔNp63α protein degradation to facilitate cancer metastasis. However, whether TAp63α is involved in TGF-ß1-induced cancer metastasis remains unclear. In this study, we show that, in human pancreatic cancer MIA PaCa-2 cells harboring p53-R248W allele, TGF-ß1 can significantly inhibit TAp63α protein stability in a Smad pathway-independent manner. Lysosome inhibitor, chloroquine, but not proteasome inhibitor MG132, can rescue TGF-ß1-induced downregulation of TAp63α protein. In addition, we show that either TGF-ß1 treatment or silencing of TAp63α can dramatically increase migration of MIA PaCa-2 cells. Importantly, the restored expression of TAp63α can effectively block TGF-ß1-induced migration of MIA PaCa-2 cells. Mechanistically, we show that TGF-ß1 promotes TAp63α protein degradation, leading to upregulation of p53-R248W protein expression, and consequently resulting in elevated MIA PaCa-2 cell migration. Together, this study indicates that lysosomal degradation is an important way for regulating TAp63α protein fate and highlights that TGF-ß1-TAp63α-mutant p53 axis is critically important in pancreatic cancer metastasis.

E47 upregulates ΔNp63α to promote growth of squamous cell carcinoma.

Targeted therapy has greatly improved both survival and prognosis of cancer patients. However, while therapeutic treatment of adenocarcinoma has been advanced greatly, progress in treatment of squamous cell carcinoma (SCC) has been slow and ineffective. Therefore, it is of great importance to decipher mechanisms and identify new drug targets involved in squamous cell carcinoma development. In this study, we demonstrate that E47 plays the distinctive and opposite roles on cell proliferation in adenocarcinoma and squamous cell carcinoma. While E47 suppresses cell proliferation in adenocarcinoma cells, it functions as a oncoprotein to promote cell proliferation and tumor growth of squamous cell carcinoma. Mechanistically, we show that E47 can directly bind to the promoter and transactivate ΔNp63 gene expression in squamous cell carcinoma cells, resulting in upregulation of cyclins D1/E1 and downregulation of p21, and thereby promoting cell proliferation and tumor growth. We further show that expression of E2A (E12/E47) is positively correlated with p63 and that high expression of E2A is associated with poor outcomes in clinical samples of squamous cell carcinoma. These results highlight that the E47-ΔNp63α axis may be potential therapeutic targets for treatment of squamous cell carcinoma.

Noncanonical TGF-ß signaling leads to FBXO3-mediated degradation of ΔNp63α promoting breast cancer metastasis and poor clinical prognosis.

Transforming growth factor-ß (TGF-ß) signaling plays a critical role in promoting epithelial-to-mesenchymal transition (EMT), cell migration, invasion, and tumor metastasis. ΔNp63α, the major isoform of p63 protein expressed in epithelial cells, is a key transcriptional regulator of cell adhesion program and functions as a critical metastasis suppressor. It has been documented that the expression of ΔNp63α is tightly controlled by oncogenic signaling and is frequently reduced in advanced cancers. However, whether TGF-ß signaling regulates ΔNp63α expression in promoting metastasis is largely unclear. In this study, we demonstrate that activation of TGF-ß signaling leads to stabilization of E3 ubiquitin ligase FBXO3, which, in turn, targets ΔNp63α for proteasomal degradation in a Smad-independent but Erk-dependent manner. Knockdown of FBXO3 or restoration of ΔNp63α expression effectively rescues TGF-ß-induced EMT, cell motility, and tumor metastasis in vitro and in vivo. Furthermore, clinical analyses reveal a significant correlation among TGF-ß receptor I (TßRI), FBXO3, and p63 protein expression and that high expression of TßRI/FBXO3 and low expression of p63 are associated with poor recurrence-free survival (RFS). Together, these results demonstrate that FBXO3 facilitates ΔNp63α degradation to empower TGF-ß signaling in promoting tumor metastasis and that the TßRI-FBXO3-ΔNp63α axis is critically important in breast cancer development and clinical prognosis. This study suggests that FBXO3 may be a potential therapeutic target for advanced breast cancer treatment.

Targeting of ΔNp63α by miR-522 promotes the migration of breast epithelial cells.

The TP63 gene, which encodes the p63 protein, is involved in multiple biological processes, including embryonic development and tumorigenesis. ΔNp63α, the predominant isoform of p63 in epithelial cells, acts as an oncogene in early-stage tumors, but paradoxically acts as a potent antimetastatic factor in advanced cancers. Here, we report that ΔNp63α is a direct target of hsa-miR-522 (miR-522). Induced expression of miR-522 reduced the levels of ΔNp63α, predisposing breast epithelial cells to a loss of epithelial and acquisition of mesenchymal morphology, resulting in accelerated collective and single-cell migration. Restoration of ΔNp63α repressed miR-522-induced migration. Interestingly, overexpression of miR-522 did not affect breast epithelial cell proliferation, suggesting that miR-522 acts specifically through ΔNp63α in this context. Furthermore, expression of miR-522-3p and p63 was negatively correlated in human cancer samples. Thus, miR-522 might be a causative factor for breast tumorigenesis and cancer metastasis. In summary, our results reveal a novel miR-522/p63 axis in cell migration and thus suggest a potential strategy for therapeutic treatment of cancer metastasis.

Hotspot mutant p53-R273H inhibits KLF6 expression to promote cell migration and tumor metastasis.

Hotspot p53 mutant proteins often gain novel functions in promoting tumor metastases. However, the molecular mechanisms by which mutant p53 exerts gain-of-function in cancer are not totally understood. In this study, we demonstrate that hotspot mutant p53, p53-R273H, promotes cell scattering growth and migration via inhibiting the expression of Krupple-like factor 6 (KLF6), a Zinc finger transcription factor and a documented tumor suppressor. Restoration of KLF6 increases the expression of E-cadherin downregulated by p53-R273H and inhibits p53-R273H-induced cell migration and tumor metastasis. Further, p53-R273H reduces KLF6 transcription by upregulating EGFR expression which in turn activates AKT-FOXO1 axis. Pharmacological inhibitor of AKT, MK2206, rescues KLF6 expression and suppresses p53-R273H-induced cell migration. Clinical analyses reveal that KLF6 expression is decreased in human breast cancer specimens harboring p53 mutations, and negatively correlated with EGFR expression in human breast cancer. In addition, low expression of KLF6 is associated with poor overall survival (OS) and relapse-free survival (RFS) in p53 mutated human breast cancer patients. Together, these results reveal an important role for EGFR-AKT-FOXO1-KLF6-E-cadherin axis in mutant p53-induced cell migration and tumor metastasis.

The Deubiquitinase USP4 Stabilizes Twist1 Protein to Promote Lung Cancer Cell Stemness.

Lung cancer stem cells (CSCs) play a pivotal role in tumor development, drug resistance, metastasis and recurrence of lung cancer. Thus, it is of great importance to study the mechanism by which CSCs are regulated. In this study, we demonstrate that the deubiquitinase USP4 is critically important in promoting lung cancer stemness. Silencing of USP4 leads to reduction of Oct4 and Sox2 expression, decreased CD133+ cell population and inhibition of tumorsphere formation. Conversely, ectopic expression of USP4 significantly enhances lung cancer cell stemness, which is effectively rescued by simultaneous silencing of Twist1. Mechanistically, we identified USP4 as a novel deubiquitinase of Twist1. USP4 binds to, deubiquitinates and stabilizes Twist1 protein. Furthermore, we show that USP4 expression is elevated in human lung cancer specimens and is positively correlated with Twist1 expression. High expression of USP4/Twist1 is associated with poor clinical outcomes of lung cancer patients. Together, this study highlights an important role for USP4 in lung cancer stemness and suggests USP4 as a potential target for lung cancer diagnosis and treatment.

Transcriptional suppression of AMPKα1 promotes breast cancer metastasis upon oncogene activation.

AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell-cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell-cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell-cell adhesion and cancer metastasis.