Barbara Muz

The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy

Hypoxia is a non-physiological level of oxygen tension, a phenomenon common in a majority of malignant tumors. Tumor-hypoxia leads to advanced but dysfunctional vascularization and acquisition of epithelial-to-mesenchymal transition phenotype resulting in cell mobility and metastasis. Hypoxia alters cancer cell metabolism and contributes to therapy resistance by inducing cell quiescence. Hypoxia stimulates a complex cell signaling network in cancer cells, including the HIF, PI3K, MAPK, and NFĸB pathways, which interact with each other causing positive and negative feedback loops and enhancing or diminishing hypoxic effects. This review provides background knowledge on the role of tumor hypoxia and the role of the HIF cell signaling involved in tumor blood vessel formation, metastasis, and development of the resistance to therapy. Better understanding of the role of hypoxia in cancer progression will open new windows for the discovery of new therapeutics targeting hypoxic tumor cells and hypoxic microenvironment.

Cell Trafficking of Endothelial Progenitor Cells in Tumor Progression

Blood vessel formation plays an essential role in many physiologic and pathologic processes, including normal tissue growth and healing, as well as tumor progression. Endothelial progenitor cells (EPC) are a subtype of stem cells with high proliferative potential that are capable of differentiating into mature endothelial cells, thus contributing to neovascularization in tumors. In response to tumor-secreted cytokines, EPCs mobilize from the bone marrow to the peripheral blood, home to the tumor site, and differentiate to mature endothelial cells and secrete proangiogenic factors to facilitate vascularization of tumors. In this review, we summarize the expression of surface markers, cytokines, receptors, adhesion molecules, proteases, and cell signaling mechanisms involved in the different steps (mobilization, homing, and differentiation) of EPC trafficking from the bone marrow to the tumor site. Understanding the biologic mechanisms of EPC cell trafficking opens a window for new therapeutic targets in cancer. Clin Cancer Res; 19(13); 3360–8. ©2013 AACR.

Spotlight on ixazomib: potential in the treatment of multiple myeloma

Despite the significant therapeutic advances achieved with proteasome inhibitors (PIs) such as bortezomib and carfilzomib in prolonging the survival of patients with multiple myeloma, the development of drug resistance, peripheral neuropathy, and pharmacokinetic limitations continue to pose major challenges when using these compounds. Ixazomib is a second-generation PI with improved activity over other PIs. Unlike bortezomib and carfilzomib, which are administered by injection, ixazomib is the first oral PI approved by US Food and Drug Administration. This review discusses the biochemical properties, mechanisms of action, preclinical efficacy, and clinical trial results leading to the US Food and Drug Administration approval of ixazomib.

Hypoxia promotes stem cell-like phenotype in multiple myeloma cells

Multiple myeloma (MM) is a plasma cell malignancy affecting the bone marrow (BM); despite the introduction of novel therapies, >90% of the MM patients relapse owing to drug resistance and microresidual disease.1 Relapse and microresidual disease in MM may be attributed to the development of a stem cell-like subpopulation, which demonstrates resistance to therapy and causes recurrence.2, 3 In solid tumors, stem cell-like cells were found to be responsible for tumor recurrence.4 Cancer stem cells are generally identified by (i) the expression of common stem cell and early-differentiation markers, (ii) G1-arrest and quiescent phenotype, (iii) capability of fast tumor initiation, (iv) chemoresistance, (v) high expression of drug transporters and enzymes detoxifying drugs; and (vi) acquisition of the epithelial-to-mesenchymal transition (EMT)-phenotype.4, 5 The stem cell-like cells were shown to adapt to hypoxic conditions to promote undifferentiated and immature phenotype of cells,6 which was associated with hypoxia-induced activation of stem cell related genes and pathways, such as Oct4, HIF, Notch, Wnt and Hedgehog.7 Similar to solid tumors, a subpopulation of MM cells was shown to demonstrate cancer stem-cell-like properties by showing clonotypic properties,3 drug resistance and high drug efflux capacity,3 and EMT-like phenotype.8 Tumor hypoxia was shown to develop during MM progression and to be crucial for metastasis.8, 9 Hypoxia was previously shown to induce the acquisition of EMT-like phenotype;8 and to promote less mature phenotype by the downregulation of plasma cell factors (IRF4, PRDM1 and XBP1) and upregulation of B-cell- (BCL6 and PAX5) and stem cell transcription factors (Oct4, NANOG, SOX2).10 CD138 (syndecan-1, a heparin sulfate proteoglycan) is the gold standard for diagnosis;11 however, previous studies showed that MM contains a subpopulation of cells that are CD138-negative, clonotypic, drug resistant, and show stem cell-like properties, express drug efflux pump,12 and display higher clonogenic potential than CD138+ in vivo.13 However, the molecular mechanisms leading to the development of this CD138-negative population and the activation of the stem-cell signaling pathways in them is not yet understood. In this study, we investigated the effect of hypoxia on the acquisition of stem cell-like properties such as early-differentiation markers, G1-arrest, quiescent phenotype, capability of tumor initiation in vivo and drug resistance in MM cells. To investigate the effect of hypoxia on the differentiation status of MM cells, five MM cell lines were cultured under normoxic (21% O2) and hypoxic conditions (in hypoxic chamber; 1% O2) for 48 h, and the expression of plasma cell marker (CD138), B-cell markers (CD19, CD20 and CD45) and hematopoietic stem cell marker (CD34) were tested. Consistently with previous studies,10 it was found that hypoxic MM cells expressed ~50% less plasma cell marker CD138 compared with normoxic conditions. On the contrary, B-cell markers such as CD20 and CD45 were increased by hypoxia, with no effect on CD19 expression. Moreover, stem cell marker CD34 was increased during hypoxia in MM cells (Figure 1a). These results confirmed that hypoxia induces immature phenotype in myeloma cells in which it decreased the expression of terminal differentiation markers (CD138) and increased the expression of B-cell and stem cell markers. Figure 1 Hypoxia promotes dedifferentiation, decreases cell proliferation, induces G1-cell cycle arrest, but not apoptosis of MM cells. The effect of hypoxia (1% O2) on (a) CD138-PerCP-Cy5.5, CD20-PE, CD45-APC, CD19-FITC and CD34-PE expression, analyzed ... We further analyzed the effect of hypoxia on cell proliferation and cell cycle in MM cells. MM cells were cultured in hypoxic and normoxic conditions, and their proliferation and associated cell signaling was analyzed. Hypoxia decreased the proliferation of MM cells by 10–20% (Figure 1b), which was associated with decreased activation of the PI3K signaling (p-PI3K-P85, p-AKT and p-mTOR), whereas stress kinases such as p-MKK3/6 and p-P38 were increased in hypoxia (Figure 1c). The reduced proliferation in hypoxia was facilitated through a G1-cell cycle arrest, with decreased synthesis of DNA (S phase) in all three cell lines (Figure 1d), which was associated with the downregulation in the expression of proteins involved in cell cycle transition from G1 to S phase, including cyclin-D1, cyclin-D2, cyclin-D3, cyclin-E and pRb, whereas the cell cycle inhibitor p27 was increased (Figure 1e). In addition, we tested the effect of hypoxia on cell apoptosis and found that both normoxic and hypoxic cells had similar percentage of apoptotic (APO 2.7-positive) cells (Figure 1f). Unchanged apoptosis was confirmed by immunoblotting showing a lack of induction of caspase-3 and caspase-9 cleavage, and unchanged expression of pro- and antiapoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1 (Figure 1g). These results suggest that hypoxia induces a quiescent state of cancer cells with no signs of cell apoptosis. Moreover, we tested the effect of hypoxia on the ability of MM1s-GFP-Luc cells to initiate tumor in vivo. MM cells were incubated in hypoxia or normoxia for 24 h in vitro and these were injected via tail vein to SCID (severe combined immunodeficiency) mice, which were followed by bioluminescence imaging for tumor progression. It was found that hypoxia enhanced the tumor initiation ability of MM cells, in which at week 3 the signal appeared first in the mice injected with hypoxic cells but not in animals injected with normoxic cells. Moreover, over the next 2 weeks the mice injected with hypoxic cells demonstrated higher tumor burden (Figures 2a and b). These findings can be attributed to the more efficient homing of hypoxic cells to the BM, as we previously described.8 Similar results were previously obtained when stem cell-like MM cells (identified as CD138-/CD34+) induced MM tumors in vivo faster and to a higher tumor burden compared with CD138+/CD34-MM cells.3 Figure 2 Hypoxia induces rapid tumor initiation and drug resistance to bortezomib and carfilzomib in MM cells. MM1s-Luc-GFP cells were incubated in normoxia or hypoxia for 24 h and injected intravenously into three severe combined immunodeficiency (SCID) ... Another important feature of stem cell-like cancer cells is drug resistance. We investigated the effect of hypoxia on MM cell proliferation in the presence of proteasome inhibitors. MM cells were cultured for 24 h in hypoxic and normoxic conditions, and subsequently treated with or without bortezomib and carfilzomib, and the proliferation was analyzed by MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay. In normoxia, bortezomib (5 nm) and carfilzomib (5 nm) exerted about 50% of killing (IC50) in tested cell lines. However, hypoxia induced complete resistance to the same concentrations of both bortezomib and carfilzomib in OPM1 cells, whereas in the case of MM1s and H929 hypoxia induced partial resistance to both drugs (Figure 2c). It has been demonstrated recently that proteasome inhibitor MG-132, led to increased reactive oxygen species (ROS) production as a potential mechanism for the induction of apoptosis in tumor cells.14 Hypoxia was shown to decrease ROS production;15, 16, 17 therefore we suggest that this could be a potential mechanism of resistance to proteasome inhibitors in hypoxia in MM cells. In general, these results on stem cell drug resistance are in accord with previous reports showing that myeloma stem cell-like cells (defined as CD138-CD19+CD27+) had an increased activity of aldehyde dehydrogenase (ALDH1), and increased the expression of drug efflux pumps such as ABCG2, ALDH1 and RARα2, conferring chemoresistance to bortezomib, dexamethasone and lenalidomide.3 Moreover, the CD138-negative subpopulation of 5T33MM and 5TGM1 cells was also more resistant to melphalan and lenalidomide than CD138+ cells.18 The hypoxic phenotype of MM cells was shown to be reversible after reoxygenation. We have previously demonstrated that hypoxic MM cells recovered, as soon as, 6 h after the exposure to medium from normoxic stroma.8 In addition, we have recently demonstrated that reoxygenation of hypoxic Waldenstroms macroglobulinemia cells reversed their hypoxic phenotype by restoring cell proliferation rate, inducing the exit of the G1-arrest, and restoring E-cadherin expression and adhesion to stromal cells.19 Moreover, the expression of the main plasma cell marker CD138, which is decreased by hypoxia, also recovered after reoxygenation in MM cells.10 In summary, hypoxia induced MM cell dedifferentiation; acquisition of a quiescent state by decreasing proliferation and inducing G1-cell cycle arrest, but without altering apoptosis; enhanced tumor initiation; and increased drug resistance to proteasome inhibitors. On the basis of these findings we propose to target hypoxic cells to diminish the stem cell-like population, in order to decrease microresidual disease and ultimately prevent recurrence in MM patients.

Molecularly Targeted Therapies in Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted.

3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma.

PURPOSE Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. EXPERIMENTAL DESIGN We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. RESULTS 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. CONCLUSIONS 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

PI3KCA plays a major role in multiple myeloma and its inhibition with BYL719 decreases proliferation, synergizes with other therapies and overcomes stroma-induced resistance.

The phosphatidylinositide 3‐kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K‐α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs.

The Role of Hypoxia and Exploitation of the Hypoxic Environment in Hematologic Malignancies

Tumor hypoxia is a well-described phenomenon during the progression of solid tumors affecting cell signaling pathways and cell metabolism; however, its role in hematologic malignancies has not been given the same attention in the literature. Therefore, this review focuses on the comparative differences between solid and hematologic malignancies with emphasis on the role of hypoxia during tumorigenesis and progression. In addition, contribution of the bone marrow and angiogenic environment are also discussed. Insight is provided into the role of hypoxia in metastatic spread, stemness, and drug resistance in hematologic conditions. Finally, emerging therapeutic strategies such as small-molecule prodrugs and hypoxia-inducible factor (HIF) targeting approaches are outlined to combat hypoxic cells and/or adaptive mechanisms in the treatment of hematologic malignancies. Mol Cancer Res; 12(10); 1347–54. ©2014 AACR.

Hypoxia Promotes Dissemination and Colonization in New Bone Marrow Niches in Waldenström Macroglobulinemia

Waldenström macroglobulinemia, a rare and indolent type of non–Hodgkin lymphoma, is characterized by widespread lymphoplasmacytic B cells in the bone marrow. Previous studies have shown that hypoxic conditions play a key role in the dissemination of other hematologic malignancies. In this study, the effect of hypoxia was tested on the progression and spread of Waldenström macroglobulinemia. Interestingly, tumor progression correlated with hypoxia levels in Waldenström macroglobulinemia cells and other cells in the bone marrow and correlated with the number of circulating tumor cells in vivo. Mechanistic studies demonstrated that hypoxia decreased cell progression and cell cycle, did not induce apoptosis, and reduced the adhesion between Waldenström macroglobulinemia cells and bone marrow stroma, through downregulation of E-cadherin expression, thus explaining increased egress of Waldenström macroglobulinemia cells to the circulation. Moreover, hypoxia increased the extravasation and homing of Waldenström macroglobulinemia cells to new bone marrow niches in vivo, by increased CXCR4/SDF-1–mediated chemotaxis and maintaining the VLA4-mediated adhesion. Re-oxygenation of hypoxic Waldenström macroglobulinemia cells enhanced the rate of proliferation and cell cycle progression and restored intercellular adhesion between Waldenström macroglobulinemia cells and bone marrow stroma. This study suggests that targeting hypoxic response is a novel strategy to prevent dissemination of Waldenström macroglobulinemia. Implications: This study provides a better understanding of the biology of dissemination of Waldenström macroglobulinemia and opens new windows for investigation of new therapeutic targets in Waldenström macroglobulinemia based on tumor hypoxia mechanisms. Mol Cancer Res; 13(2); 263–72. ©2014 AACR.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold‐standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug‐resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down‐regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138‐negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.