Arman Jahangiri

Hypoxia-Induced Autophagy Promotes Tumor Cell Survival and Adaptation to Antiangiogenic Treatment in Glioblastoma

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.

Gene Expression Profile Identifies Tyrosine Kinase c-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

Tumor Cell Autophagy as an Adaptive Response Mediating Resistance to Treatments Such as Antiangiogenic Therapy

Autophagy is a lysosomal degradation pathway that can sequester cytosolic material, including organelles, nonspecifically in a process called nonselective macroautophagy, or target specific protein aggregates designated for destruction in a process called selective autophagy. Autophagy is one mechanism that enables tumor cells to survive stressors in the tumor microenvironment, as well as injuries caused by treatments such as chemotherapy and radiation therapy. The complexity of the role of autophagy in cancer is underscored by evidence that autophagy can allow premalignant cells to escape the genotoxic stress and inflammation that promote tumorigenesis, and that some tumor cells exhibit loss of autophagy capacity altogether through molecular mechanisms that have not yet been defined. Efforts to understand and modulate the autophagy pathway will be crucial to maximize the full therapeutic potential of cancer therapies that are currently hindered by tumor cell autophagy as a resistance mechanism.

Microarray analysis verifies two distinct phenotypes of glioblastomas resistant to anti-angiogenic therapy.

Purpose: To identify mechanisms and mediators of resistance to antiangiogenic therapy in human glioblastoma. Experimental Design: We carried out microarray gene expression analysis and immunohistochemistry comparing 21 recurrent glioblastomas progressing during antiangiogenic treatment with VEGF neutralizing antibody bevacizumab to paired pretreatment tumors from the same patients. Results: Microarray analysis revealed that bevacizumab-resistant glioblastomas (BRG) had two clustering patterns defining subtypes that reflect radiographic growth patterns. Enhancing BRGs (EBRG) exhibited MRI enhancement, a long-established criterion for glioblastoma progression, and expressed mitogen-activated protein kinases, neural cell adhesion molecule-1 (NCAM-1), and aquaporin 4. Compared with their paired pretreatment tumors, EBRGs had unchanged vascularity and hypoxia, with increased proliferation. Nonenhancing BRGs (NBRG) exhibited minimal MRI enhancement but had FLAIR-bright expansion, a newer criterion for glioblastoma recurrence since the advent of antiangiogenic therapy, and expressed integrin α5, laminin, fibronectin1, and PDGFRβ. NBRGs had less vascularity, more hypoxia, and unchanged proliferation than their paired pretreatment tumors. Primary NBRG cells exhibited more stellate morphology with a 3-fold increased shape factor and were nearly 4-fold more invasive in Matrigel chambers than primary cells from EBRGs or bevacizumab-naive glioblastomas (P < 0.05). Conclusion: Using microarray analysis, we found two resistance patterns during antiangiogenic therapy with distinct molecular profiles and radiographic growth patterns. These studies provide valuable biologic insight into the resistance that has limited antiangiogenic therapy to date. Clin Cancer Res; 18(10); 2930–42. ©2012 AACR.

β1 Integrin Targeting Potentiates Antiangiogenic Therapy and Inhibits the Growth of Bevacizumab-Resistant Glioblastoma

Antiangiogenic therapies like bevacizumab offer promise for cancer treatment, but acquired resistance, which often includes an aggressive mesenchymal phenotype, can limit the use of these agents. Upregulation of β1 integrin (ITGB1) occurs in some bevacizumab-resistant glioblastomas (BRG) whereby, mediating tumor-microenvironment interactions, we hypothesized that it may mediate a mesenchymal-type resistance to antiangiogenic therapy. Immunostaining analyses of β1 integrin and its downstream effector kinase FAK revealed upregulation in 75% and 86% of BRGs, respectively, compared with pretreatment paired specimens. Furthermore, flow cytometry revealed eight-fold more β1 integrin in primary BRG cells compared with cells from bevacizumab-naïve glioblastomas (BNG). Fluorescence recovery after photobleaching of cells engineered to express a β1-GFP fusion protein indicated that the mobile β1 integrin fraction was doubled, and half-life of β1 integrin turnover in focal adhesions was reduced markedly in BRG cells compared with bevacizumab-responsive glioblastoma multiforme cells. Hypoxia, which was increased with acquisition of bevacizumab resistance, was associated with increased β1 integrin expression in cultured BNG cells. BRGs displayed an aggressive mesenchymal-like phenotype in vitro. We found that growth of BRG xenograft tumors was attenuated by the β1 antibody, OS2966, allowing a 20-fold dose reduction of bevacizumab per cycle in this model. Intracranial delivery of OS2966 through osmotic pumps over 28 days increased tumor cell apoptosis, decreased tumor cell invasiveness, and blunted the mesenchymal morphology of tumor cells. We concluded that β1 integrin upregulation in BRGs likely reflects an onset of hypoxia caused by antiangiogenic therapy, and that β1 inhibition is well tolerated in vivo as a tractable strategy to disrupt resistance to this therapy.

β1 Integrin: Critical Path to Antiangiogenic Therapy Resistance and Beyond

Angiogenesis is an important tissue-level program supporting the growth of highly aggressive cancers and early-stage metastases. However, rapid emergence of resistance to antiangiogenic therapies, such as bevacizumab, greatly limits the clinical utility of these promising approaches. The mechanisms of resistance to antiangiogenic therapy remain incompletely understood. The tumor microenvironment has been demonstrated to be a source of broad therapeutic resistance in multiple cancers. Much of the interaction between the cells comprising a tumor and their microenvironment is driven by integrins. Notably, signaling downstream of integrins in tumor cells promotes fundamental programs vital to aggressive cancer biology, including proliferation, growth, invasion, and survival signaling. These functions then can contribute to malignant phenotypes, including metastasis, therapy resistance, epithelial-to-mesenchymal transition, and angiogenesis. Accordingly, we found β1 integrin to be functionally upregulated in tumor specimens from patients after bevacizumab failure and in xenograft models of bevacizumab resistance. Inhibition of β1 in tumor cells with stable gene knockdown or treatment with OS2966, a neutralizing β1 integrin monoclonal antibody, attenuated aggressive tumor phenotypes in vitro and blocked growth of bevacizumab-resistant tumor xenografts in vivo. Thus, β1 integrins promote resistance to antiangiogenic therapy through potentiation of multiple malignant programs facilitated by interactions with the tumor microenvironment. The elucidation of this mechanism creates an outstanding opportunity for improving patient outcomes in cancer.

Hypoxia-induced tumor cell autophagy mediates resistance to anti-angiogenic therapy

While anti-angiogenic therapy was initially greeted enthusiastically by the cancer community, initial successes with this therapeutic modality were tempered by the failure of angiogenesis inhibitors to produce sustained clinical responses in most patients, with resistance to the inhibitors frequently developing. We recently reported that hypoxia increases after the devascularization caused by anti-angiogenic therapy, consistent with the goals of these therapies, but that some tumor cells become resistant and survive the hypoxic insult elicited by anti-angiogenic therapy through autophagy by activating both AMPK and HIF1A pathways. These findings suggest that modulating the autophagy pathway may someday allow anti-angiogenic therapy to fulfill its therapeutic potential. However, further work will clearly be needed to develop more potent and specific autophagy inhibitors and to better understand the regulators of autophagy in malignant cells.

Risk factors for postoperative cerebrospinal fluid leak and meningitis after expanded endoscopic endonasal surgery

Postoperative cerebrospinal fluid (CSF) leak is a serious complication of transsphenoidal surgery, which can lead to meningitis and often requires reparative surgery. We sought to identify preoperative risk factors for CSF leaks and meningitis. We reviewed 98 consecutive expanded endoscopic endonasal surgeries performed from 2008-2012 and analyzed preoperative comorbidities, intraoperative techniques, and postoperative care. Univariate and multivariate analyses were performed. The most common pathologies addressed included pituitary adenoma, Rathke cyst, chordoma, esthesioneuroblastoma, meningioma, nasopharyngeal carcinoma, and squamous cell carcinoma. There were 11 CSF leaks (11%) and 10 central nervous system (CNS) infections (10%). Univariate and multivariate analysis of preoperative risk factors showed that patients with non-ideal body mass index (BMI) were associated with higher rate of postoperative CSF leak and meningitis (both p<0.01). Also, patients with increasing age were associated with increased CSF leak (p = 0.03) and the length of time a lumbar drain was used postoperatively was associated with infection in a univariate analysis. In addition, three of three endoscopic transsphenoidal surgeries combined with open cranial surgery had a postoperative CSF leak and CNS infection rate which was a considerably higher rate than for transsphenoidal surgeries alone or surgeries staged with open cases (p<0.01 and p=0.04, respectively) In this series of expanded endoscopic transsphenoidal surgeries, preoperative BMI remains the most important preoperative predictor for CSF leak and infection. Other risk factors include age, intraoperative CSF leak, lumbar drain duration, and cranial combined cases. Risks associated with complex surgical resections when combining open and endoscopic approaches could be minimized by staging these procedures.

Suprasellar Rathke cleft cysts: clinical presentation and treatment outcomes.

BACKGROUND Rathke cleft cysts (RCCs), benign remnants of the Rathke pouch typically arising in the sella, sometimes have suprasellar extension. Purely suprasellar RCCs are rarely reported. OBJECTIVE To compare the presentations, surgical outcomes, and pathology of purely suprasellar RCCs and sellar-based RCCs. METHODS We retrospectively reviewed records, magnetic resonance images, laboratory results, and pathology of 151 RCC patients surgically managed at our institution from 1989 to 2009. The RCCs were classified as purely sellar (type I, n = 76), sellar with suprasellar extension (type II, n = 56), or purely suprasellar (type III, n = 19). RESULTS The RCCs with a suprasellar component (types II and III) more commonly presented with visual dysfunction (P < .001). Complete cyst drainage occurred in 89%, 55%, and 38% of type I, II, and III RCCs, respectively (P < .001). Vision improved in 100%, 55%, and 33% and headache improved in 74%, 64%, and 29% of type I, II, and III patients, respectively (P = .02). Temporary or permanent postoperative diabetes insipidus occurred in 5%, 16%, and 21% of type I, II, and III patients, respectively. (P < .001). In a multivariate analysis, RCC type was the only factor predicting recurrence. Kaplan-Meier 3-year recurrence/progression rates were 0%, 16%, and 29% for type I, II, and III RCCs, respectively (P < .001, type I vs II, type I vs III; P = .5 type II vs III). CONCLUSION The RCCs with a suprasellar component are neurosurgically challenging because of their proximity to the optic chiasm and infundibulum. Compared with sellar-based RCCs, RCCs with a suprasellar component more frequently present with visual dysfunction, are more difficult to completely eliminate, recur more frequently, and are associated with higher postoperative endocrine morbidity, and their preoperative visual dysfunction and headache less frequently improve with surgery. These factors must be considered during the treatment of RCCs with a suprasellar component. ABBREVIATIONS RCC: Rathke cleft cyst

A comprehensive long-term retrospective analysis of silent corticotrophic adenomas vs hormone-negative adenomas.

BACKGROUND Silent corticotrophic adenomas (SCAs) stain adrenocorticotropic hormone (ACTH)+ without causing Cushing disease. SCAs are reportedly more aggressive, but information comes from small series. OBJECTIVE To determine whether SCAs behave more aggressively than hormone-negative adenomas (HNAs), and characterize SCA ACTH production alterations. METHODS SCAs (n = 75) and HNAs (n = 1726) diagnosed at our institution from 1990 to 2011 were retrospectively reviewed. RT-PCR was used to compare expression of ACTH-producing factors. RESULTS SCA patients exhibited comparable sex and age as HNA patients (P = .7-.9). SCAs exhibited comparable size as HNAs (2.2 vs 2.0 cm, P = .2), with cavernous sinus invasion in 30% of SCAs vs 18% of HNAs (P = .03). SCA patients had higher mean preoperative serum ACTH (46 vs 19 ng/L; P = .005; normal = 5-27 ng/L), but comparable serum cortisol (13 vs 12 μg/dL; normal = 4-22 μg/dL; P < .05) as HNA patients. SCAs were gross totally resected 59% of the time, vs 53% for HNAs (P = .8). Kaplan-Meier 3-year progression/recurrence rates were 34% for strongly ACTH-positive Type I SCAs, 10% for weakly ACTH-positive Type II SCAs, and 6% for HNAs (P < .001 SCA vs HNA; P < .001 Type I vs HNA; and P = .08 Type II vs HNA). Expression of ACTH precursor pro-opiomelanocortin was 900-fold elevated in SCAs and 1300-fold elevated in Cushing disease-causing adenomas (CDCAs) vs HNAs (P < .001). Transcription of PC1/3, which cleaves pro-opiomelanocortin into ACTH, was 30-fold higher in CDCAs than SCAs (P = .02). CONCLUSION In the largest series to date, SCAs exhibited comparable size, but increased cavernous sinus invasion and progression/recurrence vs HNAs. SCAs exhibit deficient pro-opiomelanocortin to ACTH conversion. Close follow-up is warranted for SCAs.