Francesca Fontana

The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition

Proteasome inhibitors (PIs) are effective against multiple myeloma (MM), but the mechanisms of action and bases of individual susceptibility remain unclear. Recent work linked PI sensitivity to protein synthesis and proteasome activity, raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in MM cells (MMCs). Exploiting human MM lines characterized by differential PI sensitivity, we report that highly sensitive MMCs express lower proteasome levels and higher proteasomal workload than relatively PI-resistant MMCs, resulting in the accumulation of polyubiquitinated proteins at the expense of free ubiquitin (proteasome stress). Manipulating proteasome expression or workload alters apoptotic sensitivity to PI, demonstrating a cause-effect relationship between proteasome stress and apoptotic responses in MMCs. Intracellular immunostaining in primary, patient-derived MMCs reveals that polyubiquitinated proteins hallmark neoplastic plasma cells, in positive correlation with immunoglobulin (Ig) content, both intra- and interpatient. Moreover, overall proteasome activity of primary MMCs inversely correlates with apoptotic sensitivity to PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMCs to PI, potentially providing a framework for identifying indicators of responsiveness and designing novel combination therapies.

Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

Iron increases the susceptibility of multiple myeloma cells to bortezomib

Multiple myeloma is a malignant still incurable plasma cell disorder. Pharmacological treatment based on proteasome inhibition has improved patient outcome; however, bortezomib-resistance remains a major clinical problem. Inhibition of proteasome functionality affects cellular iron homeostasis and iron is a potent inducer of reactive oxygen species and cell death, unless safely stored in ferritin. We explored the potential role of iron in bortezomib-resistance. We analyzed iron proteins, oxidative status and cell viability in 7 multiple myeloma cell lines and in plasma cells from 5 patients. Cells were treated with increasing bortezomib concentrations with or without iron supplementation. We reduced ferritin levels by both shRNA technology and by drug-induced iron starvation. Multiple myeloma cell lines are characterized by distinct ferritin levels, which directly correlate with bortezomib resistance. We observed that iron supplementation upon bortezomib promotes protein oxidation and cell death, and that iron toxicity inversely correlates with basal ferritin levels. Bortezomib prevents ferritin upregulation in response to iron, thus limiting the ability to buffer reactive oxygen species. Consequently, reduction of basal ferritin levels increases both bortezomib sensitivity and iron toxicity. In patients’ cells, we confirmed that bortezomib prevents ferritin increase, that iron supplementation upon bortezomib increases cell death and that ferritin reduction overcomes bortezomib resistance. Bortezomib affects iron homeostasis, sensitizing cells to oxidative damage. Modulation of iron status is a strategy worth exploring to improve the efficacy of proteasome inhibition therapies.

Antagonizing Integrin β3 Increases Immunosuppression in Cancer.

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow–disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a “chemotherapy framing” dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. Mol Cancer Ther; 14(11); 2473–85. ©2015 AACR.

Modular prostheses in the treatment of proximal humerus metastases: review of 40 cases

BackgroundThe humerus is the second most common site of metastatic bone disease involving long bones. Tumors which have a predilection for dissemination to bone are those of breast, prostate, thyroid, lung and kidney. The rationale for surgical treatment of these lesions is to prevent or treat pathological fractures in order to relieve pain and improve function.Materials and methodsForty patients who had resection of the proximal humerus for metastatic bone disease and reconstruction with a modular prosthesis were retrospectively reviewed.ResultsMean functional outcome was 73.1% (Enneking score) and better results were achieved when a reverse prosthesis was implanted. Overall survival was 70% at 1 year, 42.5% at 2 years and 20% at 5 years. Local recurrence occurred in 4 patients, each of whom had initially been treated for a pathological fracture.ConclusionsIt is important to follow rational guidelines, like those of Capanna and Mirels, in order to prevent pathological fractures and to give the patient a definitive treatment, as the advances in the management of cancer prolong the survival of these patients. In this series, satisfactory results were obtained, giving the patients an acceptable quality of life.

N-cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage–Specific

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N‐cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N‐cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin‐insensitive Lrp5A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2‐deleted mice, suggesting N‐cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β‐catenin accumulation after administration of an anti‐Dkk1 antibody are enhanced in N‐cadherin–deficient mice. Thus, although lack of N‐cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N‐cadherin in osteolineage cells favors bone formation. Hence, N‐cadherin inhibition may widen the therapeutic window of osteoanabolic agents.

Evaluating acetate metabolism for imaging and targeting in multiple myeloma.

Purpose: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo. Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate–positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo. Experimental design: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. Results: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. Conclusions: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate–PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate–PET also detected response to therapy in vivo. Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate–PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416–29. ©2016 AACR.

Abstract 1552: Image-based microchip sorting of pure, immuno-phenotypically defined subpopulations of tumor cells from tiny formalin-fixed paraffin embedded (FFPE) samples reveals their distinct genetic features

Background: We provide a solution of pressing needs in preparation of FFPE samples for genomic analysis: small sample size, unwanted admixture of normal cells, analysis of tumor rare-cell subpopulations present at low percentages in the tumor fraction. Methods: We disaggregated into cell suspensions archival FFPE samples from 12 ovarian, pancreatic and lung cancer patients, staining for Vimentin, Keratin and DNA. We sorted by DEPArray™ precise numbers (mean = 107, median 58, range = 5-600) of pure homogenous cells from the major population of tumor cells, the contaminant diploid stromal cells, and other minority tumor cell types indicative of epithelial-to-mesenchymal transition (EMT). Using IonTorrent AmpliSeq CHPv2, we generated sequencing libraries, after lysis of the pure cells recovered by DEPArray™ (n = 54), or unsorted samples (either QIAmp DNA columns or disaggregated cells). Libraries were sequenced with IonTorrent PGM (mean depth>2,000x), and analyzed using IonTorrent software. Results: On several loci, we detected somatic mutations with 100% variant frequency, only observable as heterozygous in the unsorted samples and as wild-type in stromal cells of same patient, confirming 100% purity of sorted cells. Moreover, in the EMT-phenotype subpopulations we identified clear somatic mutations, different from tumor cells majority and undetectable in unsorted samples. Frequently, for loci harboring germ-line heterozygous SNPs with variant frequency around 50% for pure stromal cells, we readily detected loss-of-heterozygosis in tumor cells subpopulations as binary (0%/100%) variants. Quantitative traits such as copy number gains and losses were also reproducibly identified in tumor cell replicates as deviations from the 50% variant frequency of germline SNPs of pure stromal cells. Furthermore, we observed an excellent coverage uniformity (mean = 96%) for recoveries (n = 27) in the range of 81-600 cells, even higher than the uniformity obtained with (n = 2) QIAmp-purified DNA (92%). Mean uniformity gradually decreased to 89% for cell recoveries (n = 13) in the range 21-80, and further decreased to 70% for lower cell numbers (n = 14). Highlights: Sorting tumor rare-cell subpopulations reveals their genetic characteristics, undetectable in unsorted samples. Analyzing homogenous cell subpopulations boosts signal-to-noise ratio working around inherent sensitivity/specifitiy trade-offs of rare-variant calls. The proposed workflow further enables reliable detection of quantitative traits such as CNVs. Sorting pure stromal cells yields internal controls for archival samples. Citation Format: Chiara Bolognesi, Anna Doffini, Genny Buson, Rossana Lanzellotto, Giulio Signorini, Valeria Sero, Alex Calanca, Francesca Fontana, Rita Romano, Stefano Gianni, Giulia Bregola, Gianni Medoro, Raimo Tanzi, Giuseppe Giorgini, Hans Morreau, Massimo Barberis, Willem E. Corver, Nicolo Manaresi. Image-based microchip sorting of pure, immuno-phenotypically defined subpopulations of tumor cells from tiny formalin-fixed paraffin embedded (FFPE) samples reveals their distinct genetic features. [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 1552. doi:10.1158/1538-7445.AM2015-1552

Heterozygous deletion of both sclerostin (Sost) and connexin43 (Gja1) genes in mice is not sufficient to impair cortical bone modeling

Connexin43 (Cx43) is the main gap junction protein expressed in bone forming cells, where it modulates peak bone mass acquisition and cortical modeling. Genetic ablation of the Cx43 gene (Gja1) results in cortical expansion with accentuated periosteal bone formation associated with decreased expression of the Wnt inhibitor sclerostin. To determine whether sclerostin (Sost) down-regulation might contribute to periosteal expansion in Gja1 deficient bones, we took a gene interaction approach and crossed mice harboring germline null alleles for Gja1 or Sost to generate single Gja1+/–and Sost+/–and double Gja1+/–;Sost+/–heterozygous mice. In vivo μCT analysis of cortical bone at age 1 and 3 months confirmed increased thickness in Sost–/–mice, but revealed no cortical abnormalities in single Gja1+/–or Sost+/–mice. Double heterozygous Gja1+/–Sost+/–also showed no differences in mineral density, cortical thickness, width or geometry relative to wild type control mice. Likewise, 3-point bending measurement of bone strength revealed no significant differences between double Gja1+/–;Sost+/–or single heterozygous and wild type mice. Although these data do not exclude a contribution of reduced sclerostin in the cortical expansion seen in Gja1 deficient bones, they are not consistent with a strong genetic interaction between Sost and Gja1 dictating cortical modeling.