Benjamin G. Cuiffo

Huntingtin Bodies Sequester Vesicle-Associated Proteins by a Polyproline-Dependent Interaction

Polyglutamine expansion in the N terminus of huntingtin (htt) causes selective neuronal dysfunction and cell death by unknown mechanisms. Truncated htt expressed in vitro produced htt immunoreactive cytoplasmic bodies (htt bodies). The fibrillar core of the mutant htt body resisted protease treatment and contained cathepsin D, ubiquitin, and heat shock protein (HSP) 40. The shell of the htt body was composed of globules 14-34 nm in diameter and was protease sensitive. HSP70, proteasome, dynamin, and the htt binding partners htt interacting protein 1 (HIP1), SH3-containing Grb2-like protein (SH3GL3), and 14.7K-interacting protein were reduced in their normal location and redistributed to the shell. Removal of a series of prolines adjacent to the polyglutamine region in htt blocked formation of the shell of the htt body and redistribution of dynamin, HIP1, SH3GL3, and proteasome to it. Internalization of transferrin was impaired in cells that formed htt bodies. In cortical neurons of Huntingtons disease patients with early stage pathology, dynamin immunoreactivity accumulated in cytoplasmic bodies. Results suggest that accumulation of a nonfibrillar form of mutant htt in the cytoplasm contributes to neuronal dysfunction by sequestering proteins involved in vesicle trafficking.

Mesenchymal stem cells in tumor development: emerging roles and concepts.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that participate in the structural and functional maintenance of connective tissues under normal homeostasis. They also act as trophic mediators during tissue repair, generating bioactive molecules that help in tissue regeneration following injury. MSCs serve comparable roles in cases of malignancy and are becoming increasingly appreciated as critical components of the tumor microenvironment. MSCs home to developing tumors with great affinity, where they exacerbate cancer cell proliferation, motility, invasion and metastasis, foster angiogenesis, promote tumor desmoplasia and suppress anti-tumor immune responses. These multifaceted roles emerge as a product of reciprocal interactions occurring between MSCs and cancer cells and serve to alter the tumor milieu, setting into motion a dynamic co-evolution of both tumor and stromal tissues that favors tumor progression. Here, we summarize our current knowledge about the involvement of MSCs in cancer pathogenesis and review accumulating evidence that have placed them at the center of the pro-malignant tumor stroma.

Lysosomal proteases are involved in generation of N-terminal huntingtin fragments.

N-terminal mutant huntingtin (N-mhtt) fragments form inclusions and cause cell death in vitro. Mutant htt expression stimulates autophagy and increases levels of lysosomal proteases. Here, we show that lysosomal proteases, cathepsins D, B and L, affected mhtt processing and levels of cleavage products (cp) known as A and B, which form inclusions. Adding inhibitors of cathepsin D, B and L to clonal striatal cells reduced mhtt, especially mhtt fragment cp A. Mutant htt fully degraded in cathepsin-L-treated lysates but formed stable N-mhtt fragments upon exposure to cathepsin D. Mutagenesis analysis of htt cDNA suggested that cathepsin D and the protease for cp A may cleave htt in the same region. Brain lysates from HD knock-in mice expressed N-mhtt fragments that accumulated with cathepsin D treatment and declined with aspartyl protease inhibition. Findings implicate lysosomal proteases in formation of N-mhtt fragments and clearance of mhtt.

Pentraxin-3 is a PI3K signaling target that promotes stem cell-like traits in basal-like breast cancers

PI3K-stimulated induction of pentraxin-3 links innate immune signaling with the growth of basal-like breast cancer. Pentraxin-3 promotes growth of stem-like cancers The phosphoinositide 3-kinase (PI3K) pathway is activated in various cancers. Unfortunately, PI3K inhibitors have limited clinical efficacy, particularly in patients with a subtype of breast cancer characterized by cells with stem cell–like properties. Thomas et al. found that an activating mutation in the catalytic α subunit of PI3K enhanced the expression of a gene encoding pentraxin-3, a protein that functions in the innate immune response. Furthermore, an increase in the abundance of pentraxin-3 promoted stem cell–like traits in mammary epithelial and breast cancer cells. The mRNA abundance of PTX3 correlated with poor prognosis of patients with basal-like breast cancer. The findings link innate immune signaling with breast cancer development and suggest that targeting pentraxin-3 may suppress tumor growth in a subset of patients. Basal-like breast cancers (BLBCs) exhibit hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway because of the frequent mutational activation of the PIK3CA catalytic subunit and the genetic loss of its negative regulators PTEN (phosphatase and tensin homolog) and INPP4B (inositol polyphosphate-4-phosphatase type II). However, PI3K inhibitors have had limited clinical efficacy in BLBC management because of compensatory amplification of PI3K downstream signaling loops. Therefore, identification of critical PI3K mediators is paramount to the development of effective BLBC therapeutics. Using transcriptomic analysis of activated PIK3CA–expressing BLBC cells, we identified the gene encoding the humoral pattern recognition molecule pentraxin-3 (PTX3) as a critical target of oncogenic PI3K signaling. We found that PTX3 abundance is stimulated, in part, through AKT- and nuclear factor κB (NF-κB)–dependent pathways and that presence of PTX3 is necessary for PI3K-induced stem cell–like traits. We further showed that PTX3 expression is greater in tumor samples from patients with BLBC and that it is prognostic of poor patient survival. Our results thus reveal PTX3 as a newly identified PI3K-regulated biomarker and a potential therapeutic target in BLBC.

Silencing FOXP2 in breast cancer cells promotes cancer stem cell traits and metastasis.

In a recent article in Cell Stem Cell, we showed that mesenchymal stem cells (MSCs), progenitor cells that populate the breast tumor stroma, induce microRNA-mediated FOXP2 repression in breast cancer cells (BCCs), thus promoting cancer stem cell (CSC) and metastatic traits. Here, we discuss the implications of these findings for understanding metastatic CSC genesis.

Abstract 2683: The gastrointestinal microbiome and its composition are critical for antitumor efficacy of immune checkpoint inhibition by anti-PD-L1

The intestinal microbiome has become increasingly appreciated as a significant mediator of systemic antitumor immunity/response in both naive and treatment contexts. In naive contexts, an intact intestinal microbiome has been demonstrated to enhance tumorigenesis, and its composition to mediate primary tumor growth kinetics. In the context of cancer treatment, antibiotic depletion of the intestinal microbiota has been reported to inhibit the efficacy of cyclophosphamide and that of the immune checkpoint inhibitor αCTLA4. Compositional modulation of the intestinal microbiota has been found to be sufficient to enhance the antitumor efficacy of αPD-L1. Here, we assessed the relative importance of the intestinal microbiota in mediating αPD-L1 antitumor efficacy in a B16.F10.SIY murine model of melanoma, by performing parallel efficacy studies in C57BL/6 germ-free (Taconic) or specific pathogen free (Taconic or Jackson) mice. We observed that αPD-L1 treatment provided significant antitumor efficacy of in Taconic mice carrying an intact microbiome; however, this efficacy was abolished in germ-free Taconic mice. Furthermore, we observed that tumors of Jackson mice carrying an intact but compositionally different microbiome did not respond to αPD-L1 treatment. Phenotyping of local tumor and systemic immune responses, as well as characterization of the intestinal microbiome in responder vs nonresponder animals provided mechanistic insights. Taken together, these observations suggest that rational modulation of the microbiome may enhance response to immune checkpoint inhibition, and indicate that the gastrointestinal microbiome and its composition are critical for the antitumor efficacy of αPD-L1. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Katie Pedrick, Alexandra Kury, Catarina Costa, Brett Van Dam, Jonathan Jung, Gregory D. Lyng, Stephen T. Sonis. The gastrointestinal microbiome and its composition are critical for antitumor efficacy of immune checkpoint inhibition by anti-PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2017-2683

Abstract 633: Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer

Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts. Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 × 106 + 1.63 × 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 × 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays. Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX. Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Ingrid Rankin, Gregory D. Lyng, Stephano Cairo, Katie Pedrick, Alexandra Kury, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 633.

Abstract P1-07-02: Mesenchymal stem cell regulated microRNAs converge on the speech gene FOXP2 and regulate breast cancer metastasis

About 90% of breast cancer mortalities are due to the spread of breast cancer cells (BCCs) from a primary tumor to distant organs, a process known as metastasis. However, the molecular mechanisms underlying metastasis remain poorly understood. Substantial evidence now supports a major role for the tumor microenvironment (TME) in catalyzing breast cancer metastasis. Indeed, observations indicate that proximal interactions between BCCs and cells of the TME induce altered gene expression programs in BCCs, allowing for the navigation of the various steps of the metastatic cascade. Our group and others observed that breast tumors recruit mesenchymal stem cells (MSCs): multipotent fibroblasts that normally exert tissue maintenance functions. We and others have observed that physical interactions of MSCs with BCCs are sufficient to drive their metastatic dissemination in murine xenograft models, via the induction of epithelial-mesenchymal transition (EMT) and dedifferentiation into stem cell-like states (cancer stem cells, or CSCs), states tightly associated with the capacity to seed new tumors (for example in foreign tissues) and with chemotherapeutic resistance. However, the TME-induced molecular pathways regulating such mechanisms remain poorly understood. MicroRNAs (miRNAs, miRs) are small noncoding RNAs that regulate gene expression via base-pair interactions with messenger RNAs (mRNAs), resulting in mRNA degradation or translational inhibition. Due to their ability to interact with large numbers of target mRNAs simultaneously, miRNAs are major regulators of cell identity, and thereby serve critical roles in metastasis. We performed miRnome-wide screening of MSC-stimulated BCCs to determine if TME interactions might contribute to BCC metastasis via the deregulation of miRNAs. We observed that proximal MSCs induce aberrant expression of a specific set of miRNAs in BCCs, which had not been previously implicated in breast cancer pathogenesis. These miRNAs, led by the transcriptionally co-regulated miR-199a-3p and miR-214, were sufficient to actuate the metastasis of weakly metastatic human BCCs in xenograft models. We observed that exogenous expression of the miRNAs provided BCCs with phenotypes and gene markers characteristic of CSCs, including enhanced tumor initiation capacities. Interestingly, we found that the MSC-induced miRNAs function as an interrelated network, and converge upon a common novel target: the speech associated gene FOXP2. Knockdown of FOXP2 phenocopied the metastatic phenotypes observed in MSC-induced miRNA expressing BCCs. Importantly, elevated levels of the MSC-induced miRNAs or depressed levels of FOXP2 could predict patient prognosis in the clinic. Altogether, our results incriminate FOXP2 and it’s MSC-induced miRNA regulatory network as novel determinants of breast cancer metastasis. Citation Format: Benjamin G Cuiffo, Antoine Campagne, George W Bell, Antonio Lembo, Francesca Orso, Evan Lien, Manoj K Bhasin, Monica Raimo, Summer E Hanson, Andriy Marusyk, Peiman Hematti, Kornelia Polyak, Odette Mariani, Stefano Volinia, Anne Vincent-Salomon, Daniela Taverna, Antoine E Karnoub. Mesenchymal stem cell regulated microRNAs converge on the speech gene FOXP2 and regulate breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-02.

Abstract 173: Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor progression to metastasis is largely responsible for breast cancer mortalities, but the mechanisms underlying this process remain among the least understood in oncology. Cell-intrinsic mechanisms, such as accumulating genetic lesions, are well appreciated drivers of malignant progression; however, substantial evidence now supports a similarly critical role for cell-extrinsic stimuli, driving pro-malignant gene expression changes through epigenetic mechanisms. Such external stimuli derive from interactions with the tumor-associated stroma, a distinctive milieu of non-transformed stromal cells recruited to the tumor site by the growing neoplasm. Proximal interactions between cancer cells and components of this tumor-associated stroma have been demonstrated to drive angiogenesis, invasion and metastasis. Understanding these interactions will inform our knowledge of tumor progression and metastasis in general, and presents potential for developing therapeutic approaches based on interdicting the tumor-stroma crosstalk. Mesenchymal stem cells (MSCs) are ambulatory stromal progenitor cells that are normally dispatched to wound sites to aid in the repair of damaged tissues. MSCs similarly home with great affinity to tissues perturbed growing epithelial neoplasms, where they have been found to serve as stromal catalysts of carcinoma progression and metastasis in multiple experimental tumor models of a variety of human cancer contexts including lung, breast, melanoma, prostate, and colon. Despite expanding knowledge of how tumor-associated MSCs contribute to tumor malignancy, the programs and pathways deregulated in cancer cells as a consequence of their interactions with MSCs remain incompletely understood. We conducted genome-wide microRNA (miRNA) profiling analyses on MSC-exposed breast cancer cells (BCCs) to investigate how miRNA deregulation might contribute to malignant breast cancer progression. Our investigation revealed that MSCs cause aberrant expression of a distinct set of miRNAs that accelerate metastasis and are sufficient to provide cancer cells with enhanced tumor initiation capacities, including phenotypes and markers characteristic of putative cancer stem cells. We demonstrate that these miRNAs collectively constitute a regulatory network whose members converge upon a common target: the speech-associated transcriptional repressor FOXP2. FOXP2 knockdown phenocopied increases in metastasis and tumor-initiating ability of BCCs, including CSC-consistent markers, genes and phenotypes. Importantly, elevated MSC-induced miRNA levels and depressed FOXP2 levels were observed in human breast cancers as correlating with increased malignancy. Altogether, our results implicate FOXP2 and its MSC-induced miRNA regulators as novel determinants of malignant breast cancer progression. Citation Format: Benjamin Cuiffo, Antoine Campagne, George W. Bell, Evan Lien, Manoj K. Bhasin, Odette Mariani, Anne Vincent-Salomon, Antoine Karnoub. Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2014-173