Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

Overcoming hypoxia-mediated tumor progression: Combinatorial approaches targeting pH regulation, angiogenesis and immune dysfunction

Hypoxia is an important contributor to the heterogeneity of the microenvironment of solid tumors and is a significant environmental stressor that drives adaptations which are essential for the survival and metastatic capabilities of tumor cells. Critical adaptive mechanisms include altered metabolism, pH regulation, epithelial-mesenchymal transition, angiogenesis, migration/invasion, diminished response to immune cells and resistance to chemotherapy and radiation therapy. In particular, pH regulation by hypoxic tumor cells, through the modulation of cell surface molecules such as extracellular carbonic anhydrases (CAIX and CAXII) and monocarboxylate transporters (MCT-1 and MCT-4) functions to increase cancer cell survival and enhance cell invasion while also contributing to immune evasion. Indeed, CAIX is a vital regulator of hypoxia mediated tumor progression, and targeted inhibition of its function results in reduced tumor growth, metastasis, and cancer stem cell function. However, the integrated contributions of the repertoire of hypoxia-induced effectors of pH regulation for tumor survival and invasion remain to be fully explored and exploited as therapeutic avenues. For example, the clinical use of anti-angiogenic agents has identified a conundrum whereby this treatment increases hypoxia and cancer stem cell components of tumors, and accelerates metastasis. Furthermore, hypoxia results in the infiltration of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Treg) and Tumor Associated Macrophages (TAMs), and also stimulates the expression of PD-L1 on tumor cells, which collectively suppress T-cell mediated tumor cell killing. Therefore, combinatorial targeting of angiogenesis, the immune system and pH regulation in the context of hypoxia may lead to more effective strategies for curbing tumor progression and therapeutic resistance, thereby increasing therapeutic efficacy and leading to more effective strategies for the treatment of patients with aggressive cancer

Nuclear-cytoplasmic trafficking of NTF2, the nuclear import receptor for the RanGTPase, is subjected to regulation.

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s)

Cancer Therapeutic Targeting of Hypoxia Induced Carbonic Anhydrase IX: From Bench to Bedside

Carbonic Anhydrase IX (CAIX) is a major metabolic effector of tumor hypoxia and regulates intra- and extracellular pH and acidosis. Significant advances have been made recently in the development of therapeutic targeting of CAIX. These approaches include antibody-based immunotherapy, as well as use of antibodies to deliver toxic and radioactive payloads. In addition, a large number of small molecule inhibitors which inhibit the enzymatic activity of CAIX have been described. In this commentary, we highlight the current status of strategies targeting CAIX in both the pre-clinical and clinical space, and discuss future perspectives that leverage inhibition of CAIX in combination with additional targeted therapies to enable effective, durable approaches for cancer therapy.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacultyResearche

Overexpression of NTF2 restores nuclear import of Ran and nuclear export processes in Tween-20 treated HeLa cells.

<p>HeLa cells were transfected with pCMV (Empty) or pCMV-NTF2 (NTF2) for 24 h. The cells were washed and incubated in fresh serum-free DMEM containing 150 µM Tween-20 for 4 h. The cells were then washed and fixed in 1× PBS containing 4% formaldehyde. (A) The cellular location of Ran was monitored by immunofluorescence microscopy or (B) the location of tRNA^Lys was detected by FISH. (C) HeLa cells were co-transfected with NES-EGFP and pCMV or pCMV-NTF2. 24 h post-transfection, the cells were incubated in serum-free DMEM with 150 µM Tween-20 for 4 h and the cellular location of NES-EGFP was monitored by fluorescence microscopy. The nuclei were visualized by DAPI staining. T, transfected cell; NT, non-transfected cell. Scale bars represent 10 µm.</p

Tween-20 treatment causes Ran to accumulate in the cytoplasm of HeLa cells and a block in nuclear export of proteins but not nuclear import of proteins.

<p>(A) Tween-20 causes cytoplasmic retention of Ran in HeLa cells. HeLa cells were incubated in fresh serum-free DMEM (Untreated) or in serum-free DMEM containing 150 µM Tween-20 for 4 h. After the 4 h incubation, the cells were left in Tween-20 containing medium (Tween-20), washed and placed in fresh serum-free media (Wash), or had the serum-free DMEM media containing Tween-20 supplemented with 10% FBS (Serum Add) and incubated at 37°C for 1 h. The distribution of Ran was monitored by immunofluorescence microscopy as described in materials and methods. (B) Tween-20 causes nuclear accumulation of Importin-α. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 in serum-free DMEM for 4 h (Tween-20). Following the 4 h incubation, the cells were washed and incubated in serum-free DMEM for 1 h. The distribution of Importin-α was monitored by immunofluorescence microscopy. (C) Tween-20 causes nuclear accumulation of an NES-EGFP. HeLa cells were transfected with NES-EGFP and allowed to express for 24 h. Post-transfection cells were washed and placed in fresh serum-free DMEM with (Tween-20) or without (Untreated) 150 µM Tween-20 for 4 h. The distribution of NES-EGFP was monitored by direct fluorescent microscopy. (D) Tween-20 does not block nuclear import of the NLS containing Histone H2A. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 for 4 h. The distribution of Histone H2A was monitored by immunofluorescence microscopy. (E) Tween-20 does not affect TNF-α stimulated nuclear import of NF-κB. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 10 ng/ml TNF-α for 30 min, or with 150 µM Tween-20 for 4 h and then for 30 min with 10 ng/ml TNF-α. The distribution of NF-κB was monitored by immunofluorescence microscopy. The cells were DAPI stained to visualize the nucleus. Scale bar represents 10 µm.</p

The entire Tween-20 molecule is required to block nuclear tRNA export in HeLa cells.

<p>HeLa cells were treated with 50–300 µM (A) lauric acid (B), SPAN20 and (C) polyethylene glycol for 4 h in serum-free DMEM. The cells were fixed and FISH was used to monitor the location of tRNA^Lys. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

Tween-20 does not affect localization of RanGAP to the NPC.

<p>HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing 150 µM Tween-20 for 4 h (Tween-20), serum-free DMEM containing 150 µM Tween-20 for 4 h, washed and then placed in fresh serum-free DMEM for 1 h (Wash), or in serum-free DMEM containing 150 µM Tween-20 for 4 h and supplemented with 10% FBS (Serum add) and allowed to incubate for 1 h at 37°C. The cells were then washed and fixed in 1× PBS containing 4% paraformaldehyde before the distribution of RanGAP (left column) and FG repeat containing nucleoporins (mAb414, middle column) were monitored by immunofluorescence microscopy. Overlay analysis was performed to monitor any change in the localization of RanGAP (right column). Scale bar represents 10 µm.</p

Tween-80 or deoxycholate treatment does not affect nuclear tRNA export.

<p>HeLa cells were treated with (A) 150 µM Tween-80 or (B) 150 µM deoxycholate for 4 h in serum-free DMEM. The cells were fixed and FISH was used to monitor the distribution of tRNA^Lys. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

Prolonged Tween-20 treatment leads to apoptosis.

<p>HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing 150 µM Tween-20, or serum-free DMEM containing 25 µM etoposide. The cells were harvested at the time points indicated, lysed and 40 µg of cell lysate was separated by electrophoresis on a 10% SDS-PAGE gel. Western blot analysis was performed to monitor PARP cleavage.</p