Anjali Shukla

TGF-beta signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3.

CLIC4 (chloride intracellular channel 4), a multifunctional protein that traffics between the cytoplasm and nucleus, interacts with Schnurri-2, a transcription factor in the bone morphogenetic protein (BMP) signalling pathway. Here we show that transforming growth factor β (TGF-β) promotes the expression of CLIC4 and Schnurri-2 as well as their association in the cytoplasm and their translocation to the nucleus. In the absence of CLIC4 or Schnurri-2, TGF-β signalling is abrogated. Direct nuclear targeting of CLIC4 enhances TGF-β signalling and removes the requirement for Schnurri-2. Nuclear CLIC4 associates with phospho (p)-Smad2 and p-Smad3, protecting them from dephosphorylation by nuclear phosphatases. An intact TGF-β signalling pathway is essential for CLIC4-mediated growth-arrest. These results newly identify Schnurri-2 and CLIC4 as modifiers of TGF-β signalling through their stabilization of p-Smad2 and 3 in the nucleus.

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca2+-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.

CLIC4, skin homeostasis and cutaneous cancer: Surprising connections

Chloride intracellular channel 4 (CLIC4) is a putative chloride channel for intracellular organelles. CLIC4 has biological activities in addition to or because of its channel activity. In keratinocytes, CLIC4 resides in the mitochondria and cytoplasm, and CLIC4 gene expression is regulated by p53, TNF‐α, and c‐Myc. Cytoplasmic CLIC4 translocates to the nucleus in response to cellular stress conditions including DNA damage, metabolic inhibition, senescence, and exposure to certain trophic factors such as TNF‐α and LPS. Nuclear translocation is associated with growth arrest or apoptosis, depending on the level of expression. In the nucleus CLIC4 interacts with several nuclear proteins as demonstrated by yeast two‐hybrid screening and co‐immunoprecipitation. Nuclear CLIC4 appears to act on the TGF‐β pathway, and TGF‐β also causes CLIC4 nuclear translocation. In human and mouse cancer cell lines, CLIC4 levels are reduced, and CLIC4 is excluded from the nucleus. CLIC4 soluble or membrane‐inserted status is dependent on redox state, and redox alterations in cancer cells could underly the defect in nuclear translocation. CLIC4 is reduced and excluded from the nucleus of many human epithelial neoplasms. Paradoxically, CLIC4 is reciprocally upregulated in tumor stroma in conjunction with the expression of α‐smooth muscle actin in the fibroblast to myofibroblast transition. Overexpression of CLIC4 in cancer cells inhibits tumor growth in vivo. Conversely, overexpression of CLIC4 in tumor stromal cells stimulates tumor growth in vivo. Thus, CLIC4 participates in normal and pathological processes and may serve as a useful target for therapies in disturbances of homeostasis and neoplastic transformation.

CLIC4 is a tumor suppressor for cutaneous squamous cell cancer

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-β (TGF-β) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.

Cripto-1 Alters Keratinocyte Differentiation via Blockade of Transforming Growth Factor-β1 Signaling: Role in Skin Carcinogenesis

Cripto-1 is an epidermal growth factor-Cripto/FRL1/Cryptic family member that plays a role in early embryogenesis as a coreceptor for Nodal and is overexpressed in human tumors. Here we report that in the two-stage mouse skin carcinogenesis model, Cripto-1 is highly up-regulated in tumor promoter–treated normal skin and in benign papillomas. Treatment of primary mouse keratinocytes with Cripto-1 stimulated proliferation and induced expression of keratin 8 but blocked induction of the normal epidermal differentiation marker keratin 1, changes that are hallmarks of tumor progression in squamous cancer. Chemical or genetic blockade of the transforming growth factor (TGF)-β1 signaling pathway using the ALK5 kinase inhibitor SB431542 and dominant negative TGF-β type II receptor, respectively, had similar effects on keratinocyte differentiation. Our results show that Cripto-1 could block TGF-β1 receptor binding, phosphorylation of Smad2 and Smad3, TGF-β–responsive luciferase reporter activity, and TGF-β1–mediated senescence of keratinocytes. We suggest that inhibition of TGF-β1 by Cripto-1 may play an important role in altering the differentiation state of keratinocytes and promoting outgrowth of squamous tumors in the mouse epidermis. (Mol Cancer Res 2008;6(3):509–16)

Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis

Syndecan‐1 (sdc‐1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc‐1 delays wound healing, reduces migration, and increases Transforming growth factor β (TGFβ) 1 expression. In this study we show that sdc‐1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc‐1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc‐1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc‐1 null mice expressed suprabasal α3 and β4 integrins; suprabasal β4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol‐12‐myristate‐13‐acetate (TPA) did not differ among the genotypes, sdc‐1 null mice had an enhanced inflammatory response and retained higher levels of total TGFβ1 within their skin after TPA treatment. sdc‐1 null keratinocytes, transduced in vitro by oncogenic rasHa, expressed higher levels of β4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt rasHa‐transduced keratinocytes. When rasHa‐transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc‐1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc‐1 could be a useful marker for progression in neoplastic skin lesions.

An Integrated Genome-Wide Systems Genetics Screen for Breast Cancer Metastasis Susceptibility Genes

Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways in breast cancer metastasis. This analysis implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease.

Elevating CLIC4 in Multiple Cell Types Reveals a TGF-β Dependent Induction of a Dominant Negative Smad7 Splice Variant

CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling.

Abstract B05: Repurposing FDA-approved drugs for anti-metastatic therapy: Results of an integrated genome wide screen for metastasis modifiers

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastasis remains the primary cause of patient morbidity and mortality in solid tumors. Recent advances in genomic technologies have provided major insights into the etiology of tumors. However, significantly less is known about factors that contribute to metastasis. Here we report the results of a genome-wide integrated genetic, transcriptional, and epigenetic analysis in a mouse mammary tumor model to identify factors associated with tumor progression. This strategy identified a prognostic transcription signature and several upstream transcriptional regulators for metastatic disease. Interestingly, this analysis also identified novel or other FDA-approved drugs as potentially useful for anti-metastatic therapy. As predicted by this screen, administration of antidiabetic thiazolidones Rosiglitazone and Pioglitazone suppressed metastasis in an orthotopic breast cancer mouse model. Similarly, ciprofloxacin, an antibiotic, also decreased breast cancer metastasis. This strategy is thus not only of prognostic value but also presents the opportunity to rapidly repurpose drugs with proven safety and efficacy profiles for control and treatment of advanced neoplastic disease. Citation Format: Anjali Shukla, Kent W. Hunter. Repurposing FDA-approved drugs for anti-metastatic therapy: Results of an integrated genome wide screen for metastasis modifiers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B05.

Abstract IA21: An integrated genome-wide systems genetics screen for breast cancer metastasis susceptibility genes

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Hundreds or thousands of genes are thought to be associated with metastasis however how many of these genes contribute etiologically to tumor progression is not currently known. Identification of the genes contributing mechanistically to the metastatic processes will not only deepen our understanding of how metastases occur, but also provide novel targets for either preventing their formation or combatting their life-threatening effects. Previously our laboratory demonstrated that inbred mice of distinct phylogenetic lineages possess distinct propensity for metastatic disease, indicating that inherited susceptibility for metastasis exists and that polymorphisms can both mark and functionally effect genes within the metastatic cascade. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways which implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. The strategy described integrates meiotic genetic screens with epigenetic control of gene expression and three dimensional chromatin structure analyses to identify genes, molecular and cellular pathways likely to be important in metastatic disease. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease. Citation Format: Kent W. Hunter, Ling Bai, Howard H. Yang, Ying Hu, Anjali Shukla, Ngoc-Han Ha, Anthony Doran, Farhoud Faraji, Natalie Goldberger, Maxwell P. Lee, Thomas Keane. An integrated genome-wide systems genetics screen for breast cancer metastasis susceptibility genes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA21.