Nina M. Muñoz

Targeting TGF-β signaling in cancer.

Introduction: The transforming growth factor-β (TGF-β) signaling pathway has a pivotal role in tumor suppression and yet, paradoxically, in tumor promotion. Functional context dependent insights into the TGF-β pathway are crucial in developing TGF-β-based therapeutics for cancer. Areas covered: This review discusses the molecular mechanism of the TGF-β pathway and describes the different ways of tumor suppression by TGF-β. It is then explained how tumors can evade these effects and how TGF-β contributes to further growing and spreading of some of the tumors. In the last part of the review, the data on targeting TGF-β pathway for cancer treatment is assessed. This review focuses on anti-TGF-β based treatment and other options targeting activated pathways in tumors where the TGF-β tumor suppressor pathway is lost. Pre-clinical as well up to date results of the most recent clinical trials are given. Expert opinion: Targeting the TGF-β pathway can be a promising direction in cancer treatment. However, several challenges still exist, the most important are differentiating between the carcinogenic effects of TGF-β and its other physiological roles, and delineating the tumor suppressive versus the tumor promoting roles of TGF-β in each specific tumor. Future studies are needed in order to find safer and more effective TGF-β-based drugs.

TGF-β/β2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β-mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Vitamin D Deficiency Promotes Liver Tumor Growth in Transforming Growth Factor-β/Smad3-Deficient Mice Through Wnt and Toll-like Receptor 7 Pathway Modulation

Disruption of the TGF-β pathway is associated with liver fibrosis and suppression of liver tumorigenesis, conditions associated with low Vitamin D (VD) levels. However, potential contributions of VD to liver tumor progression in the context of TGF-β signaling remain unexplored. Our analyses of VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3+/− mice, with a three-fold increase in TLR7 expression compared to controls. ChIP and transcriptional assays confirm Smad3 binding at two TLR7 promoter SBE sites. Molecular interactions between TGF-β pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-β pathway member expression levels and β-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-β member expression with lower β-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-β pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and β-catenin activation. VD could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling.

Single cell lineage tracing reveals a role for TgfβR2 in intestinal stem cell dynamics and differentiation.

Significance Although Tgfβ signaling is important in intestinal development and cancer, little is known about the consequences of sporadic transforming growth factor β receptor 2 (TgfβR2) mutation in intestinal stem cells (ISCs). By labeling single, TgfβR2-mutant ISCs, we measured the effects of TgfβR2 loss on competition-driven clonal dynamics and differentiation. Specifically, we found that stochastic loss of TgfβR2 increases clonal survival while paradoxically decreasing clonal expansion and crypt fission, further elucidating mechanisms responsible for the role of Tgfβ signaling in ISCs on tumor initiation and tissue regeneration. In addition, we found that Tgfβ signaling modulates the generation of secretory cell precursors, revealing a role for Tgfβ signaling in altering ISC differentiation with implications for cancer, tissue regeneration, and inflammation. Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor β receptor 2 (TgfβR2) mutation to show cell autonomous effects of TgfβR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfβR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfβ signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfβR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfβ signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfβ signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfβ signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.

A Positive TGF-β/c-KIT Feedback Loop Drives Tumor Progression in Advanced Primary Liver Cancer

Hepatocellular carcinoma (HCC) is globally the second most common cause of cancer mortality. The majority of HCC patients are diagnosed at advanced stage disease for which no curative treatments exist. TGF-β has been identified as a potential therapeutic target. However, the molecular mechanisms mediating its functional switch from a tumor suppressor to tumor promoter in HCC and its interactions with other signaling pathways are poorly understood. Here, we demonstrate an aberrant molecular network between the TGF-β and c-KIT pathway that mediates the functional switch of TGF-β to a driver of tumor progression in HCC. TGF-β/SMAD2 signaling transcriptionally regulates expression of the c-KIT receptor ligand (stem cell factor [SCF]) with subsequent auto- and paracrine activation of c-KIT/JAK1/STAT3 signaling. SCF induces TGF-β1 ligand expression via STAT3, thereby forming a positive feedback loop between TGF-β/SMAD and SCF/c-KIT signaling. This network neutralizes TGF-β–mediated cell cycle inhibition and induces tumor cell proliferation, epithelial-to-mesenchymal-transition, migration, and invasion. Disruption of this feedback loop inhibits TGF-β tumor-promoting effects and restores its antiproliferative functions. Consistent with our in vitro data, we demonstrate SCF overexpression and its correlation to SMAD2 and STAT3 activation in human HCC tumors, advanced tumor-node-metastasis stages, and shorter survival. CONCLUSIONS: Canonical TGF-β and c-KIT signaling forms a positive, tumor-promoting feedback loop. Disruption of this loop restores TGF-β tumor suppressor function and provides the rationale for targeting the TGF-β/SCF axis as a novel therapeutic strategy for HCC.

Development of an Electroporation and Nanoparticle-based Therapeutic Platform for Bone Metastases.

Purpose To assess for nanopore formation in bone marrow cells after irreversible electroporation (IRE) and to evaluate the antitumoral effect of IRE, used alone or in combination with doxorubicin (DOX)-loaded superparamagnetic iron oxide (SPIO) nanoparticles (SPIO-DOX), in a VX2 rabbit tibial tumor model. Materials and Methods All experiments were approved by the institutional animal care and use committee. Five porcine vertebral bodies in one pig underwent intervention (IRE electrode placement without ablation [n = 1], nanoparticle injection only [n = 1], and nanoparticle injection followed by IRE [n = 3]). The animal was euthanized and the vertebrae were harvested and evaluated with scanning electron microscopy. Twelve rabbit VX2 tibial tumors were treated, three with IRE, three with SPIO-DOX, and six with SPIO-DOX plus IRE; five rabbit VX2 tibial tumors were untreated (control group). Dynamic T2*-weighted 4.7-T magnetic resonance (MR) images were obtained 9 days after inoculation and 2 hours and 5 days after treatment. Antitumor effect was expressed as the tumor growth ratio at T2*-weighted MR imaging and percentage necrosis at histologic examination. Mixed-effects linear models were used to analyze the data. Results Scanning electron microscopy demonstrated nanopores in bone marrow cells only after IRE (P , .01). Average volume of total tumor before treatment (503.1 mm3 ± 204.6) was not significantly different from those after treatment (P = .7). SPIO-DOX was identified as a reduction in signal intensity within the tumor on T2*-weighted images for up to 5 days after treatment and was related to the presence of iron. Average tumor growth ratios were 103.0% ± 75.8 with control treatment, 154.3% ± 79.7 with SPIO-DOX, 77% ± 30.8 with IRE, and -38.5% ± 24.8 with a combination of SPIO-DOX and IRE (P = .02). The percentage residual viable tumor in bone was significantly less for combination therapy compared with control (P = .02), SPIO-DOX (P , .001), and IRE (P = .03) treatment. The percentage residual viable tumor in soft tissue was significantly less with IRE (P = .005) and SPIO-DOX plus IRE (P = .005) than with SPIO-DOX. Conclusion IRE can induce nanopore formation in bone marrow cells. Tibial VX2 tumors treated with a combination of SPIO-DOX and IRE demonstrate enhanced antitumor effect as compared with individual treatments alone.

A molecular dynamics approach towards evaluating osmotic and thermal stress in the extracellular environment

Abstract Objective: A molecular dynamics approach to understanding fundamental mechanisms of combined thermal and osmotic stress induced by thermochemical ablation (TCA) is presented. Methods: Structural models of fibronectin and fibronectin bound to its integrin receptor provide idealized models for the effects of thermal and osmotic stress in the extracellular matrix. Fibronectin binding to integrin is known to facilitate cell survival. The extracellular environment produced by TCA at the lesion boundary was modelled at 37 °C and 43 °C with added sodium chloride (NaCl) concentrations (0, 40, 80, 160, and 320 mM). Atomistic simulations of solvated proteins were performed using the GROMOS96 force field and TIP3P water model. Computational results were compared with the results of viability studies of human hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B under matching thermal and osmotic experimental conditions. Results: Cell viability was inversely correlated with hyperthermal and hyperosmotic stresses. Added NaCl concentrations were correlated with a root mean square fluctuation increase of the fibronectin arginylglycylaspartic acid (RGD) binding domain. Computed interaction coefficients demonstrate preferential hydration of the protein model and are correlated with salt-induced strengthening of hydrophobic interactions. Under the combined hyperthermal and hyperosmotic stress conditions (43 °C and 320 mM added NaCl), the free energy change required for fibronectin binding to integrin was less favorable than that for binding under control conditions (37 °C and 0 mM added NaCl). Conclusion: Results quantify multiple measures of structural changes as a function of temperature increase and addition of NaCl to the solution. Correlations between cell viability and stability measures suggest that protein aggregates, non-functional proteins, and less favorable cell attachment conditions have a role in TCA-induced cell stress.

SO 2 quantification of rat HCC tumors using fluence-corrected photoacoustic imaging

Spectroscopic photoacoustic (sPA) can estimate blood oxygen saturation (SO2), which has been shown to correlate with hypoxia, and therefore could improve cancer diagnosis and treatment monitoring. However, accurate quantification of SO2 is often not straightforward as local fluence varies significantly at depth due to wavelength-dependent optical scattering and absorption. Additionally, assessing the quality of an SO2 estimate is not trivial as deep-lying (i.e., centimeter-order depth) PA data tends to be signal-limited, and SNR can vary substantially with wavelength for a particular voxel. This work implements an SNR-based thresholding approach combined with FEM-based local fluence correction to more accurately estimate SO2 in a rat model of hepatocelluar carcinoma (HCC).

Photoacoustic-ultrasonic imaging biomarkers for treatment-response assessment of sorafenib in a rat model of hepatocellular carcinoma

Sorafenib is the only clinically approved drug for hepatocellular carcinoma (HCC), but its response rate is relatively poor. Recently, it has been shown that tissue hypoxia and perfusion have predictive correlation with HCC response to sorafenib treatment. In this study, we investigate the correlation of photoacoustic-ultrasonic (PAUS) imaging biomarkers for oxygen saturation (SO2) and perfusion to the treatment response from sorafenib in an orthotopic rat model of HCC.

Abstract 892: Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Hepatocellular carcinoma (HCC) is the fifth most common tumor and the third leading cause of cancer-related deaths. Loss of TGF-β signaling has been associated with development of HCC. Cancer preventive effects of Vitamin D (VD) have been implicated in multiple cancers, however a clear role for Vitamin D in specific high risk populations remains undefined for HCC. Therefore, we examined for a potential chemopreventive role of VD in HCC in the context of TGF-β inactivation. Methods: (1) We screened for somatic mutation of the TGF-β pathway and VD related genes in 202 HCCs from The Cancer Genome Atlas (TCGA). (2) Wild type, Sptbn1+/- and Smad3+/- mice were fed with low VD (200 IU VD/kg) or high VD diet (10,000 IU VD/kg) for 9 weeks. Liver tissues were subjected to microarray analyses and further evaluation by quantitative PCR. (3) Wild type, Sptbn1+/- and Smad3+/- mice were injected Diethylnitrosamine (DEN) and at 8 month, 26 mice receiving low VD (200 IU VD/kg) and 26 mice receiving high VD (10,000 IU VD/kg) chow. HCC development was assessed at 4 months after VD treatment. (4) Reverse Phase Protein Array (RPPA) was performed to analyze expression profiles of 164 proteins of mouse liver tumors. (5) Liver samples from patients with HCV cirrhosis receiving VD supplements were examined to evaluate expression of TGF-β, Wnt and VD pathway molecules by immunohistochemistry. Results: (1) We observed a high rate of somatic mutation in TGF-β and VD pathway related genes in the TCGA genomic analysis. (2) None of the VD treated mice developed HCC but high VD treatment increased TLR7 mRNA expression about 3-fold in liver from Smad3+/- mice compared with livers from WT mice. (3) Smad3+/- mice with low VD showed 3-fold larger HCC formation, compared to the high VD group (Smad3+/-) that did not develop significant tumors. However, correction of VD in the Chow after 10 months did not reverse HCC formation. (4) RPPA data revealed that the tumor suppressor protein PDCD4 was reduced in Smad3+/- mice with low VD treatment. However, oncoproteins such as β-catenin, Stat5A and Bcl2-XL were induced in the same sample. (5) Expression of β2SP and TβRII were higher in HCV cirrhosis patients receiving VD supplementation compared to non-treated group. Conclusions: Loss of TGF-β signaling pathway developed HCC and VD deficiency promotes tumor growth in the context of Smad3 disruption potentially through regulation of TLR7 expression. However, after 10 months restoring VD does not have any significant effect on altering tumors. Therefore VD could be a potential candidate for prevention in early identified HCC high risk individuals who has inactivation of TGF-β/Smad3 signaling. Citation Format: Ji-hyun Shin, Lior H. Katz, Nina M. Munoz, Andrea Cortes, Vivek Shukla, Sang-Bae Kim, Franklin Herlong, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Jian Chen, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 892. doi:10.1158/1538-7445.AM2015-892