Thermal ablation vs. active surveillance for renal masses: a systematic review and network meta-analysis.

INTRODUCTION: Partial nephrectomy, thermal ablation and active surveillance are acceptable options for T1 stage renal tumor management. Currently, we lack sufficient information to make an accurate comparison of thermal ablation with active surveillance. The study objectives were to compare thermal ablation with active surveillance indirectly using partial nephrectomy as a reference. EVIDENCE ACQUISITION: We performed a systematic literature search using two databases (Scopus and Medline). The detailed search strategy is available at Prospero, CRD42021290055. The primary outcome was cancer-specific survival. Secondary outcomes included overall survival and metastasis-free survival. EVIDENCE SYNTHESIS: The final sample comprised 33 articles. They included the ones that compare: partial nephrectomy to ablation (29 studies), partial nephrectomy to active surveillance (2 studies), and partial nephrectomy vs. active surveillance vs. ablation (2 articles). We assessed 3-year and 5-year cancer-specific survival, and 3-, 5- and 7-year overall survival. The surface under the cumulative ranking curve (SUCRA) treatment benefit ranking was: cancer-specific survival - 48.6% for thermal ablation and 1.6% for active surveillance (5-year follow-up); overall survival - 52% for thermal ablation and 0.6% for active surveillance (7-year follow-up). The results demonstrated a significantly higher 3-year cancer-specific survival (RR 1.55, P=0.02) and 3- and 7-year follow-up overall survival (RR 1.85, P=0.03) in thermal ablation compared to active surveillance. At 5-year follow-up, cancer-specific survival and overall survival were in favor of thermal ablation while no statistically significant difference was reported. CONCLUSIONS: Thermal ablation offers a significantly higher cancer-specific survival and overall survival at mid-term follow-up in the management of T1 renal tumors compared to active surveillance. However, it is necessary to conduct further prospective randomized studies to validate the data.

Evaluating Magnetic Resonance Spectroscopy as a Tool for Monitoring Therapeutic Response of Whole Brain Radiotherapy in a Mouse Model for Breast-to-Brain Metastasis.

Brain metastases are the most common intracranial tumor in adults and are associated with poor patient prognosis and median survival of only a few months. Treatment options for brain metastasis patients remain limited and largely depend on surgical resection, radio- and/or chemotherapy. The development and pre-clinical testing of novel therapeutic strategies require reliable experimental models and diagnostic tools that closely mimic technologies that are used in the clinic and reflect histopathological and biochemical changes that distinguish tumor progression from therapeutic response. In this study, we sought to test the applicability of magnetic resonance (MR) spectroscopy in combination with MR imaging to closely monitor therapeutic efficacy in a breast-to-brain metastasis model. Given the importance of radiotherapy as the standard of care for the majority of brain metastases patients, we chose to monitor the post-irradiation response by magnetic resonance spectroscopy (MRS) in combination with MR imaging (MRI) using a 7 Tesla small animal scanner. Radiation was applied as whole brain radiotherapy (WBRT) using the image-guided Small Animal Radiation Research Platform (SARRP). Here we describe alterations in different metabolites, including creatine and N-acetylaspartate, that are characteristic for brain metastases progression and lactate, which indicates hypoxia, while choline levels remained stable. Radiotherapy resulted in normalization of metabolite levels indicating tumor stasis or regression in response to treatment. Our data indicate that the use of MR spectroscopy in addition to MRI represents a valuable tool to closely monitor not only volumetrical but also metabolic changes during tumor progression and to evaluate therapeutic efficacy of intervention strategies. Adapting the analytical technology in brain metastasis models to those used in clinical settings will increase the translational significance of experimental evaluation and thus contribute to the advancement of pre-clinical assessment of novel therapeutic strategies to improve treatment options for brain metastases patients.

The histone deacetylases HDAC1 and HDAC2 are required for the growth and survival of renal carcinoma cells.

Novel therapies are required for the treatment of metastatic renal cell carcinoma (RCC), which is associated with inoperable disease and patient death. Histone deacetylases (HDACs) are epigenetic modifiers and potential drug targets. Additional information on molecular pathways that are altered by histone deacetylase inhibitors (HDACi) in RCC cells is warranted. It should equally be delineated further which individual members of the 18 mammalian HDACs determine the survival and tumor-associated gene expression programs of such cells. Most importantly, an ongoing dispute whether HDACi promote or suppress metastasis-associated epithelial-to-mesenchymal transition (EMT) has to be resolved before HDACi are considered further as clinically relevant drugs. Here we show how HDACi affect murine and primary human RCC cells. We find that these agents induce morphological alterations resembling the metastasis-associated EMT. However, individual and proteomics-based analyses of epithelial and mesenchymal marker proteins and of EMT-associated transcription factors (EMT-TFs) reveal that HDACi do not trigger EMT. Pathway deconvolution analysis identifies reduced proliferation and apoptosis induction as key effects of HDACi. Furthermore, these drugs lead to a reduction of the cell adhesion molecule E-cadherin and of the platelet-derived growth factor receptor-ß (PDGFRß), which is a key driver of RCC metastasis formation. Accordingly, HDACi reduce the pulmonary spread of syngeneic transplanted renal carcinoma cells in mice. Specific genetic elimination of the histone deacetylases HDAC1/HDAC2 reflects the effects of pharmacological HDAC inhibition regarding growth suppression, apoptosis, and the downregulation of E-cadherin and PDGFRß. Thus, these epigenetic modifiers are non-redundant gatekeepers of cell fate and precise pharmacological targets.

Optimization of the EC26-2A4 Epitope in the gp41 Membrane Proximal External Region Targeted by Neutralizing Antibodies from an Elite Controller.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.

A Two-Phase Expansion Protocol Combining Interleukin (IL)-15 and IL-21 Improves Natural Killer Cell Proliferation and Cytotoxicity against Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children. Despite intensive research in recent decades the prognosis for patients with metastatic or relapsed diseases has hardly improved. New therapeutic concepts in anti-tumor therapy aim to modulate the patient's immune system to increase its aggressiveness or targeted effects toward tumor cells. Besides surgery, radiotherapy and chemotherapy, immune activation by direct application of cytokines, antibodies or adoptive cell therapy are promising approaches. In the last years, adoptive transfer of natural killer (NK) cells came into the focus of translational medicine, because of their high cytotoxic potential against transformed malignant cells. A main challenge of NK cell therapy is that it requires a high amount of functional NK cells. Therefore, ex vivo NK cell expansion protocols are currently being developed. Many culturing strategies are based on the addition of feeder or accessory cells, which need to be removed prior to the clinical application of the final NK cell product. In this study, we addressed feeder cell-free expansion methods using common γ-chain cytokines, especially IL-15 and IL-21. Our results demonstrated high potential of IL-15 for NK cell expansion, while IL-21 triggered NK cell maturation and functionality. Hence, we established a two-phase expansion protocol with IL-15 to induce an early NK cell expansion, followed by short exposure to IL-21 that boosted the cytotoxic activity of NK cells against RMS cells. Further functional analyses revealed enhanced degranulation and secretion of pro-inflammatory cytokines such as interferon-γ and tumor necrosis factor-α. In a proof of concept in vivo study, we also observed a therapeutic effect of adoptively transferred IL-15 expanded and IL-21 boosted NK cells in combination with image guided high precision radiation therapy using a luciferase-transduced RMS xenograft model. In summary, this two-phased feeder cell-free ex vivo culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.

Expression of the miR-302/367 cluster in glioblastoma cells suppresses tumorigenic gene expression patterns and abolishes transformation related phenotypes.

Cellular transformation is initiated by the activation of oncogenes and a closely associated developmental reprogramming of the epigenetic landscape. Transcription factors, regulators of chromatin states and microRNAs influence cell fates in development and stabilize the phenotypes of normal, differentiated cells and of cancer cells. The miR-302/367 cluster, predominantly expressed in human embryonic stem cells (hESs), can promote the cellular reprogramming of human and mouse cells and contribute to the generation of iPSC. We have used the epigenetic reprogramming potential of the miR-302/367 cluster to "de-program" tumor cells, that is, hift their gene expression pattern towards an alternative program associated with more benign cellular phenotypes. Induction of the miR-302/367 cluster in extensively mutated U87MG glioblastoma cells drastically suppressed the expression of transformation related proteins, for example, the reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC, and the transcription factors POU3F2, SALL2 and OLIG2, required for the maintenance of glioblastoma stem-like tumor propagating cells. It also diminished PI3K/AKT and STAT3 signaling, impeded colony formation in soft agar and cell migration and suppressed pro-inflammatory cytokine secretion. At the same time, the miR-302/367 cluster restored the expression of neuronal markers of differentiation. Most notably, miR-302/367 cluster expressing cells lose their ability to form tumors and to establish liver metastasis in nude mice. The induction of the miR-302/367 cluster in U87MG glioblastoma cells suppresses the expression of multiple transformation related genes, abolishes the tumor and metastasis formation potential of these cells and can potentially become a new approach for cancer therapy.

Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells.

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5.

Cytokine-regulated GADD45G induces differentiation and lineage selection in hematopoietic stem cells.

The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

STAT activation status differentiates leukemogenic from non-leukemogenic stem cells in AML and is suppressed by arsenic in t(6;9)-positive AML.

Acute myeloid leukemia (AML) is characterized by an aberrant self-renewal of hematopoietic stem cells (HSC) and a block in differentiation. The major therapeutic challenge is the characterization of the leukemic stem cell as a target for the eradication of the disease. Until now the biology of AML-associated fusion proteins (AAFPs), such as the t(15;17)-PML/RARα, t(8;21)-RUNX1/RUNX1T1 and t(6;9)-DEK/NUP214, all able to induce AML in mice, was investigated in different models and genetic backgrounds, not directly comparable to each other. To avoid the bias of different techniques and models we expressed these three AML-inducing oncogenes in an identical genetic background and compared their influence on the HSC compartment in vitro and in vivo. These AAFPs exerted differential effects on HSCs and PML/RARα, similar to DEK/NUP214, induced a leukemic phenotype from a small subpopulation of HSCs with a surface marker pattern of long-term HSC and characterized by activated STAT3 and 5. In contrast the established AML occurred from mature populations in the bone marrow. The activation of STAT5 by PML/RARα and DEK/NUP214 was confirmed in t(15;17)(PML/RARα) and t(6;9)(DEK/NUP214)-positive patients as compared to normal CD34+ cells. The activation of STAT5 was reduced upon the exposure to Arsenic which was accompanied by apoptosis in both PML/RARα- and DEK/NUP214-positive leukemic cells. These findings indicate that in AML the activation of STATs plays a decisive role in the biology of the leukemic stem cell. Furthermore we establish exposure to arsenic as a novel concept for the treatment of this high risk t(6;9)-positive AML.

The inhibition of stat5 by a Peptide aptamer ligand specific for the DNA binding domain prevents target gene transactivation and the growth of breast and prostate tumor cells.

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment.

Deacetylase inhibitors modulate proliferation and self-renewal properties of leukemic stem and progenitor cells.

Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal. The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.

Survivin inhibition by an interacting recombinant peptide, derived from the human ferritin heavy chain, impedes tumor cell growth.

BACKGROUND: Proteins involved in the aberrant regulation of signaling pathways and their downstream effectors are promising targets for cancer therapy. Survivin is an anti-apoptotic and cell cycle-promoting protein, which is consistently overexpressed in cancer cells. In normal cells, its expression is tightly controlled by signaling pathways and their associated transcriptional activators and repressors. In cancer cells, its expression is enhanced as a consequence of oncogenic signaling. We investigated the potential of a novel, peptide-based survivin inhibitor in breast cancer (SK-BR-3, MDA-MB-468) and glioblastoma (Tu9648) cells. These cells express high levels of survivin. MATERIALS AND METHODS: We downregulated survivin expression in tumor cells with a lentiviral gene transfer vector encoding a specific shRNA and a recombinant fusion protein, rSip, comprising the FTH1-derived survivin interaction domain, the human thioredoxin and a protein transduction domain. RESULTS: Downregulation of survivin expression decreased the growth and viability of tumor cells in culture and reduced growth of the cancer cells upon transplantation into immunodeficient mice. rSip selectively targets the anti-apoptotic function of survivin and causes tumor cell death. Non-transformed NIH/3T3 and MCF10A cells remain unaffected. CONCLUSIONS: rSip provides a lead structure for the development of drugs targeting the tumor cell "addiction protein" survivin.

Stat3 inhibition in neural lineage cells.

Abstract Deregulation of signal transducer and activator of transcription 3 (Stat3) is attracting attentions in neurological disorders of elderly populations, e.g., Stat3 is inactivated in hippocampal neurons of Alzheimer's disease (AD) brains, whereas it is often constitutively activated in glioblastoma multiforme (GBM), correlating with poor prognosis. Stat3-inhibiting drugs have been intensively developed for chemotherapy based on the fact that GBM, in many cases, are "addicted" to Stat3 activation. Stat3 inhibitors, however, potentially have unfavorable side effects on postmitotic neurons, normal permanent residents in the central nervous system. It is, therefore, of great importance to address detailed cellular responses of neural lineage cells including normal neurons, astrocytes, and neuronal/glial cancer cell lines to several classes of Stat3 inhibitors focusing on their effective concentrations. Here, we picked up five human and mouse cancer cell lines (Neuro-2a and SH-SY5Y neuroblastoma cell lines and Tu-9648, U-87MG, and U-373MG glioblastoma cell lines) and treated with various Stat3 inhibitors. Among them, Stattic, FLLL31, and resveratrol potently suppressed P-Stat3 and cell viability in all the tested cell lines. Stat3 knockdown or expression of dominant-negative Stat3 further sensitized cells to the inhibitors. Expression of familial AD-related mutant amyloid precursor protein sensitized neuronal cells, not glial cells, to Stat3 inhibitors by reducing P-Stat3 levels. Primary neurons and astrocytes also responded to Stat3 inhibitors with similar sensitivities to those observed in cancer cell lines. Thus, Stat3 inhibitors should be carefully targeted to GBM cells to avoid potential neurotoxicity leading to AD-like neuropsychiatric dysfunctions.

Stat3 is activated in skin lesions by the local application of imiquimod, a ligand of TLR7, and inhibited by the recombinant peptide aptamer rS3-PA.

Abstract Signal transducer and activator of transcription 3 (Stat3) assumes central functions in the regulation of apoptosis, proliferation, angiogenesis, and immune responses in normal cells. It also plays crucial roles in inflammatory and malignant diseases and in the cellular communication in the tissue microenvironment. Signaling interactions among normal endothelial cells, immune cells, and tumor cells, mediated by the release of cytokines, chemokines, and growth factors, often result in the activation of Stat3 and promotion of cancer cell proliferation, invasion, angiogenesis, and immune evasion. Stat3 also causes the differentiation and activation of T helper 17 (Th17) cells, which is involved, e.g., in psoriasis, an inflammatory autoimmune disease of the skin. Here, we describe molecular characteristics of a mouse model triggered by the treatment of mouse skin with the immune modulator imiquimod. The application of this compound causes the local release of proinflammatory cytokines and symptoms that resemble human psoriasis. We show that this process is accompanied by strong Stat3 activation. We also investigated the effects of a membrane-permeable, peptide-based Stat3 inhibitor, recombinant Stat3-specific peptide aptamer (rS3-PA). This molecule specifically interacts with Stat3 and prevents its transactivation potential in cultured cells. rS3-PA is able to penetrate the skin, enter cells, and reduce the level of activated Stat3. The topical applications of rS3-PA to the skin could thus possibly become useful in the treatment of inflammatory skin diseases and skin cancer.

Inhibition of Stat3 by peptide aptamer rS3-PA enhances growth suppressive effects of irinotecan on colorectal cancer cells.

Abstract Cytotoxic agents, alone or in combination, are being used in the treatment of colorectal cancer. Despite progress in the therapeutic regimes, this common malignancy is still the cause of considerable morbidity and mortality, and further improvements are required. Cancer cells often exhibit intrinsic resistance against chemotherapeutic agents or they develop resistance over the time of treatment. Several mechanisms have been made responsible, e.g., drugs may fail to reach tumor cells or drugs may fail to elicit cytotoxicity. The molecular characterization of drug resistance in cancer cells may lead to strategies to overcome it and enhance the sensitivity to chemotherapy. Irinotecan is one of the main treatments of colorectal cancer; it is converted into its active metabolite SN38 and acts as a topoisomerase I inhibitor. Inhibition of this enzyme prevents DNA relegation following uncoiling. Irinotecan has been used as a chemotherapeutic agent either as a single agent or in combination with 5-fluorouracil and targeted therapies directed against the epidermal growth factor receptor, such as cetuximab. The transcription factor signal transducer and activator of transcription 3 (Stat3) is a member of the signal transducer and activator of transcription protein family. Its persistent activation is found in tumor cells and has been associated with drug and radiation resistance. The treatment of colorectal cancer cells with irinotecan leads to senescence or apoptosis following DNA double-strand break induction. This process is impaired by the activation of Stat3. We have derived a Stat3 specific peptide aptamer [recombinant Stat3 inhibitory peptide aptamer (rS3-PA)] that recognizes the dimerization domain of Stat3 and effectively inhibits its function. The delivery of rS3-PA into colon cancer cells and the resulting inhibition of Stat3 strongly enhanced the cytotoxic action of SN38. These data show that the targeted inhibition of Stat3 decreases drug resistance and enhances SN38-mediated cell death. The combination of these agents has a potent antitumor effect and could become beneficial for the treatment of patients with colorectal cancer.

A membrane penetrating peptide aptamer inhibits STAT3 function and suppresses the growth of STAT3 addicted tumor cells.

Cancer cells are characterized by the aberrant activation of signaling pathways governing proliferation, survival, angiogenesis, migration and immune evasion. These processes are partially regulated by the transcription factor STAT3. This factor is inappropriately activated in diverse tumor types. Since tumor cells can become dependent on its persistent activation, STAT3 is a favorable drug target. Here, we describe the functional characterization of the recombinant STAT3 inhibitor, rS3-PA. This inhibitor is based on a 20 amino acid peptide which specifically interacts with the dimerization domain of STAT3. It is integrated into a thioredoxin scaffold and fused to a protein transduction domain. Protein gel blot and immunofluorescence analyses showed that rS3-PA is efficiently taken up by cells via an endocytosis independent mechanism. Intracellularly, it reduces the phosphorylation of STAT3 and enhances its degradation. This leads to the downregulation of STAT3 target gene expression on the mRNA and protein levels. Subsequently, tumor cell proliferation, survival and migration and the induction of angiogenesis are inhibited. In contrast, normal cells remain unaffected. Systemic administration of rS3-PA at doses of 7.5 mg/kg reduced P-STAT3 levels and significantly inhibited tumor growth up to 35% in a glioblastoma xenograft mouse model.

Tracking the activation of Stat5 through the expression of an inducible reporter gene in a transgenic mouse line.

Signal transducer and activator of transcription 5 (Stat5), a latent cytoplasmic transcription factor, becomes activated by phosphorylation upon cytokine, hormone, and growth factor interactions with their appropriate receptors and induces the transcription of target genes. It plays crucial roles in principal cell fate decisions and regulates cell differentiation, development, proliferation, apoptosis, and inflammation. It is active in the mammary gland, the liver, hematopoietic cells, and other organs and has pleiotropic functions, depending on its activation pathway and its site of action. We derived transgenic mice in which the expression of a LacZ reporter gene is directed by Stat5-specific response elements and visualized the activation of Stat5 in cells of mouse organs at different developmental stages. The reporter gene activity reflects the timing and the location of Stat5 activation and was documented in mammary epithelial cells during developmental stages of the gland, cells of the liver, kidney, spleen, thymus, and uterus and in granulocytes and macrophages of the transgenic lines.

The integration of a Stat3 specific peptide aptamer into the thioredoxin scaffold protein strongly enhances its inhibitory potency.

We are characterizing peptides which are able to interact with functional domains of oncoproteins and thus inhibit their activity. The yeast two-hybrid system was used to derive a peptide sequence which specifically interacts with the dimerization domain of the transcription factor Stat3. The activated form of Stat3 is required for the survival of many transformed cells and Stat3 inhibition can cause tumor cell death. The genetic selection of specific peptide sequences from random peptide libraries requires the integration into a scaffold protein and the expression in yeast cells. The scaffold protein, a variant of the human thioredoxin protein, has previously been optimized and also allows for effective bacterial expression of the recombinant protein and the cellular uptake of the purified, recombinant protein. We investigated the contributions of the scaffold protein to the inhibitory properties of rS3-PA. For this purpose we compared rS3-PA in which the ligand peptide is embedded within the thioredoxin scaffold protein with a minimal Stat3-interacting peptide sequence. sS3-P45 is a synthetic peptide of 45 amino acids in length and consists only of the Stat3-binding sequence of 20 amino acids, a protein transduction domain (PTD) and a Flag-tag. Both, the recombinant rS3-PA of 19.3 kDa and the synthetic sS3-P45 of 5.1 kDa, were taken up into the cytoplasm of cells by the PTD-mediated transduction process, inhibited Stat3 target gene expression and caused the death of Stat3-dependent tumor cells. Stat3-independent normal cells were unaffected. rS3-PA effectively inhibited Stat3 function at 2 µM, however, sS3-P45 was required at a concentration of 100 µM to exert the same effects. The more potent action of rS3-PA is most probably due to a conformational stabilization of the Stat3-interacting peptide in the context of the scaffold protein.

The intracellular delivery of a recombinant peptide derived from the acidic domain of PIAS3 inhibits STAT3 transactivation and induces tumor cell death.

Signaling components, which confer an "addiction" phenotype on cancer cells, represent promising drug targets. The transcription factor signal transducers and activators of transcription 3 (STAT3) is constitutively activated in many different types of tumor cells and its activity is indispensible in a large fraction. We found that the expression of the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3 (PIAS3), positively correlates with STAT3 activation in normal cells. This suggests that PIAS3 controls the extent and the duration of STAT3 activity in normal cells and thus prevents its oncogenic function. In cancer cells, however, the expression of PIAS3 is posttranscriptionally suppressed, possibly enhancing the oncogenic effects of activated STAT3. We delimited the interacting domains of STAT3 and PIAS3 and identified a short fragment of the COOH-terminal acidic region of PIAS3, which binds strongly to the coiled-coil domain of STAT3. This PIAS3 fragment was used to derive the recombinant STAT3-specific inhibitor rPP-C8. The addition of a protein transduction domain allowed the efficient internalization of rPP-C8 into cancer cells. This resulted in the suppression of STAT3 target gene expression, in the inhibition of migration and proliferation, and in the induction of apoptosis at low concentrations [half maximal effective concentration (EC(50)), <3 micromol/L]. rPP-C8 did not affect normal fibroblasts and represents an interesting lead for the development of novel cancer drugs targeting the coiled-coil domain of STAT3.

Stem cells of the breast and cancer therapy.

Breast cancer remains a significant public health problem despite advances in the understanding of the molecular and cellular events that underlie the disease. Crucial pathways regulating the cell cycle, proliferation and survival of breast cancer cells have been investigated and aberrant components of these pathways have been exploited as new drug targets. However, the mortality from breast cancer is only slowly declining. Recently, a model has been proposed that might explain the heterogeneous biological features of breast cancer cell populations and their differential response to therapeutic agents, which has interesting implications for further progress in therapy. This model links the emergence of breast cancer cells to stem cells and progenitors, an observation originally made in other cancer entities. It hypothesizes that the tumors originate from a small population of undifferentiated cells. These cells can undergo self-renewal and are able to generate a large number of partially differentiated cells, which constitute the bulk of the tumor. These cancer stem cells resemble adult stem and progenitor cells found in the normal breast, but are deregulated in their patterns of proliferation and differentiation. They could originate from normal stem cells or from more differentiated progenitors and lose their normal growth restraints through a series of oncogenic mutations that deregulate a small number of central signaling pathways. If breast cancer really is a stem and progenitor cell disease, this will have important implications for the understanding of the emergence of cancer cells. A combination of the cell-type of origin, stem cells, early or late progenitors and the particular oncogenic mutations acquired could provide a new classification of the different types of breast cancer. These parameters might determine the mechanisms of cancer progression and the responsiveness of patients to drug treatment. Stem cell-specific features could possibly be exploited as innovative drug targets.