Fibroblast growth factor receptor 1 inhibition suppresses pancreatic cancer chemoresistance and chemotherapy-driven aggressiveness.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often intrinsically-resistant to standard-of-care chemotherapies such as gemcitabine. Acquired gemcitabine resistance (GemR) can arise from treatment of initially-sensitive tumors, and chemotherapy can increase tumor aggressiveness. We investigated the molecular mechanisms of chemoresistance and chemotherapy-driven tumor aggressiveness, which are understood incompletely. METHODS: Differential proteomic analysis was employed to investigate chemotherapy-driven chemoresistance drivers and responses of PDAC cells and patient-derived tumor xenografts (PDX) having different chemosensitivities. We also investigated the prognostic value of FGFR1 expression in the efficacy of selective pan-FGFR inhibitor (FGFRi)-gemcitabine combinations. RESULTS: Quantitative proteomic analysis of a highly-GemR cell line revealed fibroblast growth factor receptor 1 (FGFR1) as the highest-expressed receptor tyrosine kinase. FGFR1 knockdown or FGFRi co-treatment enhanced gemcitabine efficacy and decreased GemR marker expression, implicating FGFR1 in augmentation of GemR. FGFRi treatment reduced PDX tumor progression and prolonged survival significantly, even in highly-resistant tumors in which neither single-agent showed efficacy. Gemcitabine exacerbated aggressiveness of highly-GemR tumors, based upon proliferation and metastatic markers. Combining FGFRi with gemcitabine or gemcitabine+nab-paclitaxel reversed tumor aggressiveness and progression, and prolonged survival significantly. In multiple PDAC PDXs, FGFR1 expression correlated with intrinsic tumor gemcitabine sensitivity. CONCLUSION: FGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse.

Expression of fibroblast growth factor receptor 1 correlates inversely with the efficacy of single-agent fibroblast growth factor receptor-specific inhibitors in pancreatic cancer.

BACKGROUND AND PURPOSE: Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. EXPERIMENTAL APPROACH: The mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. RESULTS: The pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. CONCLUSIONS AND IMPLICATIONS: Single-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1.

PAK1 and Therapy Resistance in Melanoma.

Malignant melanoma claims more lives than any other skin malignancy. While primary melanomas are usually cured via surgical excision, the metastatic form of the disease portents a poor prognosis. Decades of intense research has yielded an extensive armamentarium of anti-melanoma therapies, ranging from genotoxic chemo- and radiotherapies to targeted interventions in specific signaling pathways and immune functions. Unfortunately, even the most up-to-date embodiments of these therapies are not curative for the majority of metastatic melanoma patients, and the need to improve their efficacy is widely recognized. Here, we review the reports that implicate p21-regulated kinase 1 (PAK1) and PAK1-related pathways in the response of melanoma to various therapeutic modalities. Ample data suggest that PAK1 may decrease cell sensitivity to programmed cell death, provide additional stimulation to growth-promoting molecular pathways, and contribute to the creation of an immunosuppressive tumor microenvironment. Accordingly, there is mounting evidence that the concomitant inhibition of PAK1 enhances the potency of various anti-melanoma regimens. Overall, the available information suggests that a safe and effective inhibition of PAK1-dependent molecular processes would enhance the potency of the currently available anti-melanoma treatments, although considerable challenges in implementing such strategies still exist.

The Development of Nonthermal Plasma and Tirapazamine as a Novel Combination Therapy to Treat Melanoma In Situ.

Although melanoma accounts for only 5.3% of skin cancer, it results in >75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ.

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Proteotoxic Stress as an Exploitable Vulnerability in Cells with Hyperactive AKT.

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.

Regulation of local GTP availability controls RAC1 activity and cell invasion.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

The role of polo-like kinase 3 in the response of BRAF-mutant cells to targeted anticancer therapies.

The activation of oncogenic mitogen-activated protein kinase cascade via mutations in BRAF is often observed in human melanomas. Targeted inhibitors of BRAF (BRAFi), alone or as a part of a combination therapy, offer a significant benefit to such patients. Unfortunately, some cases are initially nonresponsive to these drugs, while others become refractory in the course of treatment, underscoring the need to understand and mitigate the underlying resistance mechanisms. We report that interference with polo-like kinase 3 (PLK3) reduces the tolerance of BRAF-mutant melanoma cells to BRAFi, while increased PLK3 expression has the opposite effect. Accordingly, PLK3 expression correlates with tolerance to BRAFi in a panel of BRAF-mutant cell lines and is elevated in a subset of recurring BRAFi-resistant melanomas. In PLK3-expressing cells, R406, a kinase inhibitor whose targets include PLK3, recapitulates the sensitizing effects of genetic PLK3 inhibitors. The findings support a role for PLK3 as a predictor of BRAFi efficacy and suggest suppression of PLK3 as a way to improve the efficacy of targeted therapy.

The role of PAK1 in the sensitivity of kidney epithelial cells to ischemia-like conditions.

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury.

KLF9-dependent ROS regulate melanoma progression in stage-specific manner.

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten-/- primary melanomas. It also, as expected, promoted BrafCA/Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

XBP1-KLF9 Axis Acts as a Molecular Rheostat to Control the Transition from Adaptive to Cytotoxic Unfolded Protein Response.

Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress.

Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma.

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

FOXQ1 controls the induced differentiation of melanocytic cells.

Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAFV600E-dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

The evidence for a microRNA product of human DROSHA gene.

MicroRNAs are short RNA molecules that regulate function and stability of a large subset of eukaryotic mRNAs. In the main pathway of microRNA biogenesis, a short "hairpin" is excised from a primary transcript by ribonuclease DROSHA, followed by additional nucleolytic processing by DICER and inclusion of the mature microRNA into the RNA-induced silencing complex. We report that a microRNA-like molecule is encoded by human DROSHA gene within a predicted stem-loop element of the respective transcript. This putative mature microRNA is complementary to DROSHA transcript variant 1 and can attenuate expression of the corresponding protein. The findings suggest a possibility for a negative feedback loop, wherein DROSHA processes its own transcript and produces an inhibitor of its own biosynthesis.

P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells.

BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms.

MicroRNA analysis suggests an additional level of feedback regulation in the NF-κB signaling cascade.

It is increasingly clear that the biological functions of a transcription factor cannot be fully understood solely on the basis of protein-coding genes that fall under its control. Many transcription factors regulate expression of miRNAs, which affect the cell by modulating translation and stability of mRNAs. The identities and the roles of NF-κB-regulated miRNAs have been attracting research interest for a long time. We revisited this issue in a system with controlled expression of one of the key regulators of NF-κB, RIPK1. Several regulated miRNAs were identified, including miR-146a, miR-215 and miR-497. The miRNAs were also inducible by IL-1ß, but not when NF-κB activity was repressed by mutant IκBα. The presence of a miR-497 site was predicted in the 3'-UTR of IKBKB gene, which encodes IKKß. Using appropriately engineered reporters, we confirmed that this site can be a target of suppressive action of miR-497. Our findings suggest that NF-κB controls expression of a miRNA, which may reduce production of IKKß. Considering the role of IKKß in the canonical pathway of NF-κB activation, our observations may indicate a new mechanism that modulates the magnitude of such activation, as well as the propensity of a cell to engage canonical vs. non-canonical pathways.

Protein kinase A type II-α regulatory subunit regulates the response of prostate cancer cells to taxane treatment.

In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6-22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy.