Walden Ai

γ-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia

Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2–like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.Summary Gamma-secretase inhibitors (GSIs) block the activation of oncogenic NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor auto-up-regulation and induced apoptotic cell death through induction of BIM expression. GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of Klf4, a negative regulator of cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of Ccnd2 and protected mice from developing intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1, side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell–like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b + Ly6G + immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b+Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b+Ly6G+ cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+ IMCs in early cancer development.

KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.

MicroRNA-155 deficiency enhances the recruitment and functions of myeloid-derived suppressor cells in tumor microenvironment and promotes solid tumor growth

Immune cells in tumor microenvironment play a prominent role in tumor progression and metastasis. MicroRNA‐155 (miR‐155) represents an important player in innate and adaptive immunity by regulating differentiation, maturation and activation of macrophages, dendritic cells, B cells and T cells. However, the role of miR‐155 expression in immune cells in solid tumor development is less elucidated. Our current study showed that both B16‐F10 melanoma and Lewis lung carcinoma tumors grew much faster in bic/miR‐155 knockout (miR‐155−/−) mice along with an increase of myeloid‐derived suppressor cells (MDSCs) accumulation in tumors, compared to that in wild‐type mice. Bone marrow transplantation study showed that bone marrow miR‐155 deficiency could replicate the above tumor‐promoting phenotype. In vitro study demonstrated that tumor‐infiltrating miR‐155−/− MDSCs showed greater migration ability and expressed higher level of multiple chemokines. Furthermore, we found that the level of HIF‐1α, a direct target of miR‐155, was increased in miR‐155 deficient MDSCs, and that the increased HIF‐1α upregulated CXCL1, CXCL3 and CXCL8 expression in MDSCs, contributing to the enhanced recruitment of miR‐155−/− MDSCs to the tumors. Moreover, miR‐155−/− MDSCs showed enhanced immunosuppressive and pro‐angiogenic capacities. Taken together, our study, for the first time, demonstrated that miR‐155 deficiency promoted solid tumor growth through increasing the recruitment of MDSCs to tumor microenvironment and enhancing the tumor‐promoting functions of the recruited MDSCs. Thus, upregulating miR‐155 expression in MDSCs may be developed as a therapeutic approach to halt tumor development.

Deficiency of the Kruppel-like factor KLF4 correlates with increased cell proliferation and enhanced skin tumorigenesis.

Kruppel-like factor 4 (KLF4) is a transcription factor that is highly expressed in differentiated epithelial cells including that of the skin. It is critical for specification or function of differentiated epithelial cells. Moreover, KLF4 functions either as a tumor suppressor or an oncogene depending on different cellular contexts. However, the role of KLF4 in skin tumorigenesis remains controversial. To address this issue, we first examined KLF4 expression using a cohort of samples from patients with skin squamous cell carcinoma and basal cell carcinoma and found that in 21 of 24 tumor tissues (87.5%), KLF4 expression as assayed by immunohistochemistry was absent when compared with that in normal tissues. In addition, knockdown of KLF4 in human epidermal squamous cell carcinoma SCC13 cells was accompanied by increased cell growth. Further analysis revealed that KLF4 deficiency promoted cell migration and adhesion, which are the important properties of tumor cells. These observations were supported by the effect upon overexpression of KLF4 in SCC13 cells. Furthermore, we generated a novel tamoxifen-inducible KLF4/CreER and KLF4(flox) double transgenic mouse model to examine the role of KLF4 in skin cancer development. Consistent with in vitro studies, KLF4 deficiency increased the ability of migration and adhesion of mouse primary skin keratinocytes. Moreover, KLF4 knockout led to increased cell proliferation and skin carcinogenesis in a classical DMBA/TPA mouse skin cancer model. Taken together, our data suggest that KLF4 inhibits cell proliferation, migration and adhesion and that loss of KLF4 promotes skin tumorigenesis.

Krüppel-like factor 4 regulates intestinal epithelial cell morphology and polarity.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.

Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity

Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OSWt), expression of dnMaml1 at the ocular surface (OSdnMaml1) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OSdnMaml1 phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OSdnMaml1 dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.

miR-155–Deficient Bone Marrow Promotes Tumor Metastasis

Infiltration of immune cells in primary tumors and metastatic sites is known to influence tumor progression and metastasis. Macrophages represent the most abundant immune cells in the tumor microenvironment, and evidence has shown that macrophages promote seeding, extravasation, and persistent growth of tumor cells at metastatic sites. miR-155 plays an essential role in immune cell development/function, and its aberrant expression is associated with lymphomas and several solid tumor types. However, it is unknown how miR-155 expression in immune cells affects solid tumor growth and metastasis. To this end, bone marrow transplantation was performed using miR-155–deficient mice as bone marrow donors and wild-type (WT) mice as recipients, and the chimeric mice were inoculated with tumor cells. We demonstrate that bone marrow lacking miR-155 significantly enhanced lung metastasis without a substantial effect on primary tumor growth. Relative to mice with WT bone marrow, miR-155–deficient bone marrow accumulated more macrophages in the spleen and lungs. Further analysis revealed that miR-155–deficient macrophages in metastatic sites exhibited a tumor-promoting M2 phenotype. In vitro study suggested that miR-155–null macrophages were prone to M2 polarization upon incubation with tumor cell–conditioned medium, due to elevated expression of C/EBPβ, an identified miR-155 target. These data, for the first time, demonstrate that miR-155 in host immune cells plays a vital role in modulating solid tumor metastasis by affecting the recruitment and polarization of bone marrow–derived macrophages. Implications: Targeted inhibition of miR-155 delays tumor development but inhibition in host immune cells may encourage metastasis. Mol Cancer Res; 11(8); 923–36. ©2013 AACR.

Deficiency of Kruppel-like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid-derived suppressor cells

Increasing evidence indicates that myeloid‐derived suppressor cells (MDSCs) negatively regulate immune responses during tumor progression, inflammation and infection. However, the underlying molecular mechanisms of their development and mobilization remain to be fully delineated. Kruppel‐like factor KLF4 is a transcription factor that has an oncogenic function in breast cancer development, but its function in tumor microenvironment, a critical component for tumorigenesis, has not been examined. By using a spontaneously metastatic 4T1 breast cancer mouse model and an immunodeficient NOD/SCID mouse model, we demonstrated that KLF4 knockdown delayed tumor development and inhibited pulmonary metastasis, which accompanied by decreased accumulation of MDSCs in bone marrow, spleens and primary tumors. Mechanistically, we found that KLF4 knockdown resulted in a significant decrease of circulating GM‐CSF, an important cytokine for MDSC biology. Consistently, recombinant GM‐CSF restored the frequency of MDSCs in purified bone marrow cells incubated with conditioned medium from KLF4 deficient cells. In addition, we identified CXCL5 as a critical mediator to enhance the expression and function of GM‐CSF. Reduced CXCL5 expression by KLF4 knockdown in primary tumors and breast cancer cells was correlated with a decreased GM‐CSF expression in our mouse models. Finally, we found that CXCL5/CXCR2 axis facilitated MDSC migration and that anti‐GM‐CSF antibodies neutralized CXCL5‐induced accumulation of MDSCs. Taken together, our data suggest that KLF4 modulates maintenance of MDSCs in bone marrow by inducing GM‐CSF production via CXCL5 and regulates recruitment of MDSCs into the primary tumors through the CXCL5/CXCR2 axis, both of which contribute to KLF4‐mediated mammary tumor development.