ZBTB7B is a permissive regulator of hepatocellular carcinoma initiation by repressing c-Jun expression and function.

Hepatocarcinogenesis is a multi-step process. However, the regulators of hepatocellular carcinoma (HCC) initiation are understudied. Adult liver-specific gene expression was globally downregulated in HCC. We hypothesize that adult liver-specific genes, especially adult liver-enriched transcription factors may exert tumor-suppressive functions in HCC. In this study, we identify ZBTB7B, an adult liver-enriched transcription factor as a permissive regulator of HCC initiation. ZBTB7B is highly expressed in hepatocytes in adult livers, compared to fetal livers. To evaluate the functions of ZBTB7B in hepatocarcinogenesis, we performed hepatocyte-specific ZBTB7B knockout in hydrodynamic oncogene transfer-induced mouse liver cancer models. Hepatocyte-specific knockout of ZBTB7B promotes activated Akt and N-Ras-induced HCC development. Moreover, ZBTB7B deficiency sensitizes hepatocytes to a single oncogene Akt-induced oncogenic transformation and HCC initiation, which is otherwise incompetent in inducing HCC. ZBTB7B deficiency accelerates HCC initiation by down-regulating adult liver-specific gene expression and priming livers to a fetal-like state. The molecular mechanism underlying ZBTB7B functions in hepatocytes was investigated by integrated transcriptomic, phosphoproteomic, and chromatin immunoprecipitation-sequencing analyses. Integrative multi-omics analyses identify c-Jun as the core signaling node in ZBTB7B-deficient liver cancer initiation. c-Jun is a direct target of ZBTB7B essential to accelerated liver cancer initiation in ZBTB7B-deficient livers. Knockdown of c-Jun expression or dominant negative c-Jun expression delays HCC development in ZBTB7B-deficient livers. In addition, ZBTB7B competes with c-Jun for chromatin binding. Ectopic ZBTB7B expression attenuates the tumor-promoting functions of c-Jun. Expression of ZBTB7B signature, composed of 140 genes co-regulated by ZBTB7B and c-Jun, is significantly downregulated in early-stage HCCs compared to adjacent normal tissues, correlates to liver-specific gene expression, and is associated with good prognosis in human HCC. Thus, ZBTB7B functions as a permissive regulator of HCC initiation by directly regulating c-Jun expression and function.

Intact regulation of G1/S transition renders esophageal squamous cell carcinoma sensitive to PI3Kα inhibitors.

Phosphatidylinositol 3-kinase alpha (PI3Kα) inhibitors are currently evaluated for the therapy of esophageal squamous cell carcinoma (ESCC). It is of great importance to identify potential biomarkers to predict or monitor the efficacy of PI3Kα inhibitors in an aim to improve the clinical responsive rate in ESCC. Here, ESCC PDXs with CCND1 amplification were found to be more sensitive to CYH33, a novel PI3Kα-selective inhibitor currently in clinical trials for the treatment of advanced solid tumors including ESCC. Elevated level of cyclin D1, p21 and Rb was found in CYH33-sensitive ESCC cells compared to those in resistant cells. CYH33 significantly arrested sensitive cells but not resistant cells at G1 phase, which was associated with accumulation of p21 and suppression of Rb phosphorylation by CDK4/6 and CDK2. Hypo-phosphorylation of Rb attenuated the transcriptional activation of SKP2 by E2F1, which in turn hindered SKP2-mediated degradation of p21 and reinforced accumulation of p21. Moreover, CDK4/6 inhibitors sensitized resistant ESCC cells and PDXs to CYH33. These findings provided mechanistic rationale to evaluate PI3Kα inhibitors in ESCC patients harboring amplified CCND1 and the combined regimen with CDK4/6 inhibitors in ESCC with proficient Rb.

Type IV collagen α5 chain promotes luminal breast cancer progression through c-Myc-driven glycolysis.

Cancer cell metabolism reprogramming is one of the hallmarks of cancer. Cancer cells preferentially utilize aerobic glycolysis, which is regulated by activated oncogenes and the tumor microenvironment. Extracellular matrix (ECM) in the tumor microenvironment, including the basement membranes (BMs), is dynamically remodeled. However, whether and how ECM regulates tumor glycolysis is largely unknown. We show that type IV collagens, components of BMs essential for the tissue integrity and proper function, are differentially expressed in breast cancer subtypes that α5 chain (α5(IV)) is preferentially expressed in the luminal-type breast cancer and is regulated by estrogen receptor-α. α5(IV) is indispensable for luminal breast cancer development. Ablation of α5(IV) significantly reduces the growth of luminal-type breast cancer cells and impedes the development of luminal-type breast cancer. Impaired cell growth and tumor development capability of α5(IV)-ablated luminal breast cancer cells is attributed to the reduced expression of glucose transporter and glycolytic enzymes and impaired glycolysis in luminal breast cancer cells. Non-integrin collagen receptor discoidin domain receptor-1 (DDR1) expression and p38 mitogen-activated protein kinase activation are attenuated in α5(IV)-ablated luminal breast cancer cells, resulting in reduced c-Myc oncogene expression and phosphorylation. Ectopic expression of constitutively active DDR1 or c-Myc restores the expression of glucose transporter and glycolytic enzymes, and thereafter restores aerobic glycolysis, cell proliferation, and tumor growth of luminal breast cancer. Thus, type IV collagen α5 chain is a luminal-type breast cancer-specific microenvironmental regulator modulating cancer cell metabolism.

Autocrine pro-legumain promotes breast cancer metastasis via binding to integrin αvß3.

Tumor metastasis is the leading cause of cancer-associated mortality. Unfortunately, the underlying mechanism of metastasis is poorly understood. Expression of legumain (LGMN), an endo-lysosomal cysteine protease, positively correlates with breast cancer metastatic progression and poor prognosis. Here, we report that LGMN is secreted in the zymogen form by motile breast cancer cells. Through binding to cell surface integrin αvß3 via an RGD motif, the autocrine pro-LGMN activates FAK-Src-RhoA signaling in cancer cells and promotes cancer cell migration and invasion independent of LGMN protease activity. Either silencing LGMN expression or mutationally abolishing pro-LGMN‒αvß3 interaction significantly inhibits cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Finally, we developed a monoclonal antibody against LGMN RGD motif, which blocks pro-LGMN‒αvß3 binding, and effectively suppresses cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Thus, disruption of pro-LGMN‒integrin αvß3 interaction may be a potentially promising strategy for treating breast cancer metastasis.

Perivenous Stellate Cells Are the Main Source of Myofibroblasts and Cancer-Associated Fibroblasts Formed After Chronic Liver Injuries.

BACKGROUND AND AIMS: Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. APPROACH AND RESULTS: Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. CONCLUSIONS: In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-ß signaling links HSC activation to liver fibrosis and tumorigenesis.

The micropeptide LEMP plays an evolutionarily conserved role in myogenesis.

In recent years, micropeptides have been increasingly identified as important regulators in various biological processes. However, whether micropeptides are functionally conserved remains largely unknown. Here, we uncovered a micropeptide with evolutionarily conserved roles in myogenesis. RNA-seq data analysis of proliferating mouse satellite cells (SCs) and differentiated myotubes identified a previously annotated lncRNA, MyolncR4 (1500011K16RIK), which is upregulated during muscle differentiation. Significantly, MyolncR4 is highly conserved across vertebrate species. Multiple lines of evidence demonstrate that MyolncR4 encodes a 56-aa micropeptide, which was named as LEMP (lncRNA encoded micropeptide). LEMP promotes muscle formation and regeneration in mouse. In zebrafish, MyolncR4 is enriched in developing somites and elimination of LEMP results in impaired muscle development, which could be efficiently rescued by expression of the mouse LEMP. Interestingly, LEMP is localized at both the plasma membrane and mitochondria, and associated with multiple mitochondrial proteins, suggestive of its involvement in mitochondrial functions. Together, our work uncovers a micropeptide that plays an evolutionarily conserved role in skeletal muscle differentiation, pinpointing the functional importance of this growing family of small peptides.

Protein C receptor is a therapeutic stem cell target in a distinct group of breast cancers.

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr+ cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR+ TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

α3 Chains of type V collagen regulate breast tumour growth via glypican-1.

Pericellular α3(V) collagen can affect the functioning of cells, such as adipocytes and pancreatic ß cells. Here we show that α3(V) chains are an abundant product of normal mammary gland basal cells, and that α3(V) ablation in a mouse mammary tumour model inhibits mammary tumour progression by reducing the proliferative potential of tumour cells. These effects are shown to be primarily cell autonomous, from loss of α3(V) chains normally produced by tumour cells, in which they affect growth by enhancing the ability of cell surface proteoglycan glypican-1 to act as a co-receptor for FGF2. Thus, a mechanism is presented for microenvironmental influence on tumour growth. α3(V) chains are produced in both basal-like and luminal human breast tumours, and its expression levels are tightly coupled with those of glypican-1 across breast cancer types. Evidence indicates α3(V) chains as potential targets for inhibiting tumour growth and as markers of oncogenic transformation.

C/EBPß Mediates TNF-α-Induced Cancer Cell Migration by Inducing MMP Expression Dependent on p38 MAPK.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine that triggers cell proliferation, cell death, or inflammation. Besides its cytotoxic effect on cancer cells, TNF-α exerts tumor promoting activity. Aberrant TNF-α signaling promotes cancer cell motility, invasiveness, and enhances cancer metastasis. Exaggerated tumor cell migration, invasion, and metastasis by TNF-α has been attributed to the activation of NF-κB signaling. It is yet to be elucidated if other signaling pathways and effector molecules are involved in TNF-α-induced cancer cell migration and metastasis. Expression of C/EBPß, a transcription factor involved in metabolism, inflammation, and cancer, is increased upon TNF-α treatment. TNF-α induces C/EBPß expression by enhancing its transcription and protein stability. Activation of p38 MAPK, but not NF-κB or JNK, is responsible for TNF-α-induced stabilization of C/EBPß protein. C/EBPß is involved in TNF-α-induced cancer cell migration. Knockdown of C/EBPß inhibits TNF-α-induced cell migration, while overexpression of C/EBPß increases migration of cancer cells. C/EBPß is translated into transcriptional activator LAP1 and LAP2 and transcriptional repressor LIP utilizing alternative in-frame translation start sites. Despite TNF-α induces expression of all three isoforms, LAP1/2, but not LIP, promote cancer cell migration. TNF-α induced MMP1/3 expression, which was abrogated by C/EBPß knockdown or p38 MAPK inhibition. MMP inhibitor or knockdown of MMP1/3 diminished TNF-α- and C/EBPß-induced cell migration. Thus, C/EBPß mediates TNF-α-induced cancer cell migration by inducing MMP1/3 expression, and may participate in the regulation of inflammation-associated cancer metastasis.

Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1.

Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of KrasG12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV.

Lysyl oxidase promotes bleomycin-induced lung fibrosis through modulating inflammation.

Enzymes involved in collagen biosynthesis, including lysyl oxidase (LOX), have been proposed as potential therapeutic targets for idiopathic pulmonary fibrosis. LOX expression is significantly upregulated in bleomycin (BLM)-induced lung fibrosis, and knockdown of LOX expression or inhibition of LOX activity alleviates the lung fibrosis. Unexpectedly, treatment of the mice with LOX inhibitor at the inflammatory stage, but not the fibrogenic stage, efficiently reduces collagen deposition and normalizes lung architecture. Inhibition of LOX impairs inflammatory cell infiltration, TGF-ß signaling, and myofibroblast accumulation. Furthermore, ectopic expression of LOX sensitizes the fibrosis-resistant Balb/c mice to BLM-induced inflammation and lung fibrosis. These results suggest that LOX is indispensable for the progression of BLM-induced experimental lung fibrosis by aggravating the inflammatory response and subsequent fibrosis process after lung injury.

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma.

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

Activator protein 1 suppresses antitumor T-cell function via the induction of programmed death 1.

T cells play a critical role in tumor immunosurveillance by eliminating newly transformed somatic cells. However, tumor cell variants can escape from immunological control after immunoediting, leading to tumor progression. Whether and how T cells respond to tumor growth remain unclear. Here, we found that tumor-infiltrating T cells exhibited persistently up-regulated expression of the activator protein 1 (AP-1) subunit c-Fos during tumor progression. The ectopic expression of c-Fos in T cells exacerbated tumor growth, whereas the T-cell-specific deletion of c-Fos reduced tumor malignancy. This unexpected immunosuppressive effect of c-Fos was mediated through the induced expression of immune inhibitory receptor programmed death 1 (PD-1) via the direct binding of c-Fos to the AP-1-binding site in the Pdcd1 (gene encoding PD-1) promoter. A knock-in mutation of this binding site abrogated PD-1 induction, augmented antitumor T-cell function and repressed tumor growth. Taken together, these findings indicate that T-cell c-Fos subsequently induces PD-1 expression in response to tumor progression and that disrupting such induction is essential for repression of tumor growth.

Lysyl oxidase, extracellular matrix remodeling and cancer metastasis.

Lysyl oxidase (LOX) family oxidases, LOX and LOXL1-4, oxidize lysine residues in collagens and elastin, resulting in the covalent crosslinking and stabilization of these extracellular matrix (ECM) structural components, thus provide collagen and elastic fibers much of their tensile strength and structural integrity. Abnormality in LOX expression and/or activity results in connective tissue disorders and fibrotic diseases. Despite LOX family oxidases have been reported to function as tumor suppressors, recent studies have highlighted the roles of LOX family oxidases in promoting cancer metastasis. LOX family oxidases are highly expressed in invasive tumors, and are closely associated with metastasis and poor patient outcome. Consistent to their roles in connective tissue homeostasis, LOX family oxidases expedite tumorigenesis and metastasis through active remodeling of tumor microenvironment. LOX family oxidases are also actively involved in the process of epithelial-mesenchymal transition (EMT), an event critical in cancer cell invasion and metastasis. In this review, we will summarize the recent progress on LOX family oxidases, with much of the focus on the roles and mechanism of LOX in tumor progression and metastasis.

LKB1 in lung cancerigenesis: a serine/threonine kinase as tumor suppressor.

Lung cancer is featured with high mortality, with a 15% five-year survival rate worldwide. Genetic alterations, such as loss of function of tumor suppressor genes, frequently contribute to lung cancer initiation, progression and metastasis. Liver kinase B1 (LKB1), as a serine/threonine kinase and tumor suppressor, is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Recent studies have provided strong evidences that LKB1 loss promotes lung cancerigenesis process, especially lung cancer progression and metastasis. This review will summarize recent progress on how LKB1 modulates the process of lung cancerigenesis, emphasizing on LKB1 downstream signaling pathways and biological functions. We will further discuss the potential development of prognostic biomarkers or therapeutic targets in lung cancer clinic based on the molecular alteration associated with deregulated LKB1 signaling.