Combination of proteasome and HDAC inhibitor enhances HPV16 E7-specific CD8+ T cell immune response and antitumor effects in a preclinical cervical cancer model

Background

Bortezomib, a proteasome inhibitor and suberoylanilide hydroxamic acid (SAHA, also known as Vorinostat), a histone deacetylase inhibitor, have been recognized as potent chemotherapeutic drugs. Bortezomib and SAHA are FDA-approved for the treatment of cutaneous T cell lymphoma and multiple myeloma/mantle cell lymphoma, respectively. Furthermore, the combination of the bortezomib and SAHA has been tested in a variety of preclinical models and in clinical trials and may be ideal for the treatment of cancer. However, it remains unclear how this treatment strategy affects the host immune response against tumors.

Results

Here, we used a well-defined E6/E7-expressing tumor model to examine how the immune system can be motivated to act against tumor cells expressing tumor antigens. We demonstrate that the combination of bortezomib and SAHA elicits potent antitumor effects in TC-1 tumor-bearing mice. Additionally, we are the first to show that treatment with bortezomib and SAHA leads to tumor-specific immunity by rendering tumor cells more susceptible to killing by antigen-specific CD8+ T cells than treatment with either drug alone.

Conclusions

The current study serves an important foundation for the future clinical application of both drugs for the treatment of cervical cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0111-1) contains supplementary material, which is available to authorized users.     

Non-Invasive Imaging Provides Spatiotemporal Information on Disease Progression and Response to Therapy in a Murine Model of Multiple Myeloma

Background

Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.

Material and Methods

A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.

Results

Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase⁺ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase⁺ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.

Conclusions

This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.     

Proteasome inhibitor bortezomib ameliorates intestinal injury in mice

Background

Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis.

Methodology/Principal Findings

Bortezomib treatment was shown to potently inhibit murine DSS-induced colitis. The attenuation of DSS-induced colitis was associated with decreased inflammatory cell infiltration in the colon. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4⁺ and CD8⁺ T cells in the colon and mesenteric lymph nodes. Bortezomib treatment significantly diminished interferon (IFN)-γ expression in the colon and mesenteric lymph nodes. Furthermore, cytoplasmic IFN-γ production by CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes was substantially decreased by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting nuclear factor-κB activation during DSS-induced colitis.

Conclusions/Significance

Bortezomib treatment is likely to induce T cell death, thereby suppressing DSS-induced colitis by reducing IFN-γ production.     

Variation in Drug Sensitivity of Malignant Mesothelioma Cell Lines with Substantial Effects of Selenite and Bortezomib,Highlights Need for Individualized Therapy

Background

Malignant mesothelioma cells have an epithelioid or sarcomatoid morphology, both of which may be present in the same tumor. The sarcomatoid phenotype is associated with worse prognosis and heterogeneity of mesothelioma cells may contribute to therapy resistance, which is often seen in mesothelioma. This study aimed to investigate differences in sensitivity between mesothelioma cell lines to anti-cancer drugs. We studied two novel drugs, selenite and bortezomib and compared their effect to four conventional drugs. We also investigated the immunoreactivity of potential predictive markers for drug sensitivity; Pgp, MRP-1, ERCC1, RRM1, TS, xCT and proteasome 20S subunit.

Materials and methods

We treated six mesothelioma cell lines with selenite, bortezomib, carboplatin, pemetrexed, doxorubicin or gemcitabine as single agents and in combinations. Viability was measured after 24 and 48 hours. Immunocytochemistry was used to detect predictive markers.

Results

As a single agent, selenite was effective on four out of six cell lines, and in combination with bortezomib yielded the greatest response in the studied mesothelioma cell lines. Cells with an epithelioid phenotype were generally more sensitive to the different drugs than the sarcomatoid cells. Extensive S-phase arrest was seen in pemetrexed-sensitive cell lines. MRP-1 predicted sensitivity of cell lines to treatment with carboplatin and xCT predicted pemetrexed effect.

Conclusions

The observed heterogeneity in sensitivity of mesothelioma cell lines with different morphology highlights the need for more individualized therapy, requiring development of methods to predict drug sensitivity of individual tumors. Selenite and bortezomib showed a superior effect compared to conventional drugs, motivating clinical testing of these agents as future treatment regime components for patients with malignant mesothelioma.     

The Use of Novel Drugs Can Effectively Improve Response,Delay Relapse and Enhance Overall Survival in Multiple Myeloma Patients with Renal Impairment

Background

Renal impairment is a common feature in multiple myeloma and is considered a poor prognostic factor.

Aim

To determine the impact of novel drugs (i.e. bortezomib, lenalidomide and thalidomide) in the treatment of myeloma patients with renal impairment. The primary endpoint was overall survival and secondary endpoints were time to next treatment and response.

Methods

The study population included all patients diagnosed with treatment-demanding multiple myeloma January 2000 to June 2011 at 15 Swedish hospitals. Renal impairment was defined as an estimated glomerular filtration rate under 60 mL/min/1.73 m².

Result

The study population consisted of 1538 patients, of which 680 had renal impairment at diagnosis. The median overall survival in patients with renal impairment was 33 months, which was significantly shorter than 52 months in patients with normal renal function (P<0.001). Novel agents in first line improved overall survival (median 60 months) in non-high-dose treated patients with renal impairment (n = 143) as compared to those treated with conventional cytotoxic drugs (n = 411) (median 27 months) (P<0.001). In the multivariate analysis up front treatment with bortezomib was an independent factor for better overall survival in non-high-dose treated renally impaired patients. High-dose treated renally impaired patients had significantly better median overall survival than non-high-dose ones (74 versus 26 months) and novel drugs did not significantly improve survival further in these patients. Patients with renal impairment had both a shorter median time to next treatment and a lower response rate than those with normal renal function. However, novel drugs and high dose treatment lead to a significantly longer time to next treatment and the use of novel agents significantly improved the response rate of these patients.

Conclusion

High dose treatment and novel drugs, especially bortezomib, can effectively overcome the negative impact of renal impairment in patients with multiple myeloma.     

Ser1333 phosphorylation indicates ROCKI activation

Background

Two isoforms of Rho-associated protein kinase (ROCK), ROCKI and ROCKII, play a pivotal role in regulation of cytoskeleton and are involved in multiple cellular processes in mammalian cells. Knockout mice experiments have indicated that the functions of ROCKI and II are probably non-redundant in physiology. However, it is difficult to differentiate the activation status of ROCKI and ROCKII in biological samples. Previously, we have identified phosphorylation site of ROCKII at Ser1366 residue sensitive to ROCK inhibition. We further investigated the activity-dependent phosphorylation site in ROCKI to establish the reagents that can be used to detect their individual activation.

Results

The phosphorylation site of ROCKI sensitive to its inhibition was identified to be the Ser1333 residue. The ROCKI pSer1333-specific antibody does not cross-react with phosphorylated ROCKII. The extent of S1333 phosphorylation of ROCKI correlates with myosin II light chain phosphorylation in cells in response to RhoA stimulation.

Conclusions

Active ROCKI is phosphorylated at Ser1333 site. Antibodies that recognize phospho-Ser1333 of ROCKI and phospho-S1366 residues of ROCKII offer a means to discriminate their individual active status in cells and tissues.     

Small compound 6-O-angeloylplenolin induces mitotic arrest and exhibits therapeutic potentials in multiple myeloma

Background

Multiple myeloma (MM) is a disease of cell cycle dysregulation while cell cycle modulation can be a target for MM therapy. In this study we investigated the effects and mechanisms of action of a sesquiterpene lactone 6-O-angeloylplenolin (6-OAP) on MM cells.

Methodology/Principal Findings

MM cells were exposed to 6-OAP and cell cycle distribution were analyzed. The role for cyclin B1 to play in 6-OAP-caused mitotic arrest was tested by specific siRNA analyses in U266 cells. MM.1S cells co-incubated with interleukin-6 (IL-6), insulin-like growth factor-I (IGF-I), or bone marrow stromal cells (BMSCs) were treated with 6-OAP. The effects of 6-OAP plus other drugs on MM.1S cells were evaluated. The in vivo therapeutic efficacy and pharmacokinetic features of 6-OAP were tested in nude mice bearing U266 cells and Sprague-Dawley rats, respectively. We found that 6-OAP suppressed the proliferation of dexamethasone-sensitive and dexamethasone-resistant cell lines and primary CD138+ MM cells. 6-OAP caused mitotic arrest, accompanied by activation of spindle assembly checkpoint and blockage of ubiquitiniation and subsequent proteasomal degradation of cyclin B1. Combined use of 6-OAP and bortezomib induced potentiated cytotoxicity with inactivation of ERK1/2 and activation of JNK1/2 and Casp-8/-3. 6-OAP overcame the protective effects of IL-6 and IGF-I on MM cells through inhibition of Jak2/Stat3 and Akt, respectively. 6-OAP inhibited BMSCs-facilitated MM cell expansion and TNF-α-induced NF-κB signal. Moreover, 6-OAP exhibited potent anti-MM activity in nude mice and favorable pharmacokinetics in rats.

Conclusions/Significance

These results indicate that 6-OAP is a new cell cycle inhibitor which shows therapeutic potentials for MM.     

Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

Background

T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known.

Methodology/Principal Findings

Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin.

Conclusions/Significance

We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.     

Regulation of stathmin phosphorylation in mouse liver progenitor‐29 cells during proteasome inhibition

Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor‐induced apoptosis. We have investigated the response of mouse liver progenitor‐29 (MLP‐29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC‐MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor‐induced apoptosis in MLP‐29 cells. Particularly, a transient modification of the phosphorylation state of Ser¹⁶, Ser²⁵ and Ser³⁸, which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin‐activated protein kinase II, extracellular regulated kinase‐1/2 and cyclin‐dependent kinase‐2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.     

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens

Background

There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.

Results

Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.

Conclusion

In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.     

Cellular Contractility Requires Ubiquitin Mediated Proteolysis

Background

Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.

Principal Findings

We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.

Conclusions

Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.     

Expression of Neuron-Specific Enolase in Multiple Myeloma and Implications for Clinical Diagnosis and Treatment

Objective

To determine the expression of neuron-specific enolase (NSE) in patients with multiple myeloma (MM) and to evaluate its clinical value as a tumor marker and, an indicator of disease progression and treatment efficacy.

Methods

Using electrochemiluminescence immunoassay (ECLIA), we measured the serum levels of NSE in 47 healthy subjects (control group), 25 patients with small cell lung cancer (lung cancer group), and 52 patients with MM (MM group). For the MM group, serum NSE levels were measured and other disease indicators and related symptoms were monitored before and after chemotherapy. The relationship between NSE expression and other MM-related factors was analyzed. In addition, immunohistochemical staining was performed on bone marrow biopsy specimens from patients with MM.

Results

In the control group, serum NSE levels were within the normal range as previously reported, while the lung cancer group and the untreated MM group exhibited NSE levels that were significantly higher relative to the control group (P<0.05). The difference in NSE expression between the lung cancer group and untreated MM group was statistically significant (P<0.05). NSE levels were significantly decreased in MM patients after chemotherapy and were positively correlated with an MM disease index [beta-2 microglobulin (β₂-MG)]. Changes in NSE were not related to the response rate to chemotherapy but rather were correlated with progression-free survival.

Conclusions

Patients with MM may have increased serum NSE levels, and changes in NSE may provide insight into treatment efficacy of chemotherapy and disease progression. Perhaps NSE expression is a viable biomarker for MM and can be a useful reference for the design and adjustment of clinical MM treatment programs.     

Subcutaneous Bortezomib in Multiple Myeloma Patients Induces Similar Therapeutic Response Rates as Intravenous Application But It Does Not Reduce the Incidence of Peripheral Neuropathy

Objective

Subcutaneous (SC) application of bortezomib has been recently introduced as a new application route in multiple myeloma (MM) patients. We performed an analysis to compare the outcomes of bortezomib-based therapy in multiple myeloma (MM) patients treated using either intravenous (IV) or subcutaneous (SC) route of administration.

Patients and methods

During January 2012 through December 2013, we performed a retrospective analysis of 446 patients with MM treated with bortezomib-based regimens (either once weekly – 63% or twice weekly – 27%) in both, the first line setting, and in relapse, with separate analysis of patients undergoing autologous stem cell transplantation. We assessed the response rates and toxicity profiles in both, IV and SC route of bortezomib administration.

Results

The response rates in both IV and SC arm were similar with overall response rate 71.7% vs 70.7%, complete remissions in 13.9% vs 8.6%, very good partial remissions in 30.8% vs 34.5% and partial remissions in 27% vs 27.6%. The most frequent grade ≥3 toxicities were anemia, thrombocytopenia and neutropenia, with no significant differences between IV and SC group. There were no significant differences in the rate of peripheral neuropathy (PN). PN of any grade was present in 48% in the IV arm and in 41% in the SC arm. PN grade ≥2 was present in 20% vs 18% and PN grade ≥3 was present in 6% vs 4%.

Conclusions

We conclude that subcutaneous application of bortezomib has similar therapeutic outcomes and toxicity profile as intravenous route of application. In our cohort there was no difference in the incidence of PN, suggesting that PN is dose dependent and might be reduced by lower intensity schemes rather than by the route of administration.