Marie Grøn Saelen

Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma

BackgroundThe histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials.MethodsRadiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models.ResultsUnder hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth.ConclusionsVorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials.

Controlling systolic blood pressure is difficult in patients with diabetic kidney disease exhibiting moderate-to-severe reductions in renal function.

This study compared the use of antihypertensive treatment and blood pressure (BP) controls between patients with diabetic kidney disease (DK+) and patients with non‐diabetic kidney disease (DK−) exhibiting moderate‐to‐severe chronic renal failure who did not need renal replacement therapy. A cross‐sectional survey included all renal patients with s‐creatinine at ⩾200 µmol/l attending regular control sessions at six renal units in Norway. Of the 351 patients included, 73 (20.8%) were DK+. The proportion reaching a BP goal of <130/80 mmHg was similar in DK+ and DK− (14.1% vs 13.6%, p = 0.92), while 38% and 39% achieved a BP of <140/90 mmHg, respectively. The systolic BP goal was more difficult to achieve than the diastolic BP goal in DK+ patients (35% vs 15%) despite a mean of three different types of drugs being used. Loop diuretics and beta‐adrenergic‐receptor antagonists were the most frequently prescribed drugs, and the use of angiotensin‐converting enzyme inhibitors or angiotensin‐II‐receptor antagonists declined when renal function deteriorated, from 80% to 0% and from 66% to 20% in the DK+ and DK− groups, respectively (p = 0.001). Thus, despite the use of multiple antihypertensive drugs, controlling BP – especially the systolic BP – is difficult in high‐risk patients with chronic renal failure caused by diabetic kidney disease.

Tumor kinase activity in locally advanced rectal cancer: Angiogenic signaling and early systemic dissemination

Tumor hypoxia is a common determinant of resistance to cytotoxic therapies and metastatic behavior. In rectal cancer patients receiving preoperative chemoradiotherapy, tyrosine kinase activities in tumors with poor and good treatment responses were found to differ. Given that tyrosine kinase signaling mediates hypoxic tissue adaptation, the present study examined whether tumor kinase activity might also correlate with systemic dissemination of rectal cancer. Immunomagnetic selection of disseminated tumor cells (DTC) from bone marrow aspirates was undertaken in 55 patients with locally advanced rectal cancer. Using peptide arrays with 144 tyrosine kinase substrates, phosphopeptide signatures were generated from patients’ baseline tumor biopsies, to study association between DTC and tumor tyrosine kinase activity regulated ex vivo by sunitinib. Disseminated tumor cells were detected in 60% of cases, and these patients had significantly poorer metastasis-free survival than patients without DTC. Phosphorylation of 31 array tyrosine kinase substrates by tumor samples was significantly more strongly inhibited by sunitinib in the DTC-negative patients, with a number of phosphosubstrates representing angiogenic factors. In this cohort of rectal cancer patients, tumor phenotypes defined by a subset of tyrosine kinase activities correlating with weak ex vivo inhibition by sunitinib, was associated with early systemic dissemination.

Hypoxic tumor kinase signaling mediated by STAT5A in development of castration-resistant prostate cancer.

In this study, we hypothesized that androgen-deprivation therapy (ADT) in prostate cancer, although initially efficient, induces changes in the tumor kinome, which subsequently promote development of castration-resistant (CR) disease. Recognizing the correlation between tumor hypoxia and poor prognosis in prostate cancer, we further hypothesized that such changes might be influenced by hypoxia. Microarrays with 144 kinase peptide substrates were applied to analyze CWR22 prostate carcinoma xenograft samples from ADT-naïve, androgen-deprived (AD), long-term AD (ADL), and CR disease stages. The impact of hypoxia was assessed by matching the xenograft kinase activity profiles with those acquired from hypoxic and normoxic prostate carcinoma cell cultures, whereas the clinical relevance was evaluated by analyzing prostatectomy tumor samples from patients with locally advanced disease, either in ADT-naïve or early CR disease stages. By using this novel peptide substrate microarray method we revealed high kinase activity mediated by signal transducer and activator of transcription 5A (STAT5A) in CR prostate cancer. Additionally, we uncovered high STAT5A kinase activity already in regressing ADL xenografts, before renewed CR growth was evidenced. Finally, since increased STAT5A kinase activity also was detected after exposing prostate carcinoma cells to hypoxia, we propose long-term ADT to induce tumor hypoxia and stimulate STAT5A kinase activity, subsequently leading to renewed CR tumor growth. Hence, the study detected STAT5A as a candidate to be further investigated for its potential as marker of advanced prostate cancer and as possible therapeutic target protein.

Tumor Phosphatidylinositol-3-Kinase Signaling and Development of Metastatic Disease in Locally Advanced Rectal Cancer

Background Recognizing EGFR as key orchestrator of the metastatic process in colorectal cancer, but also the substantial heterogeneity of responses to anti-EGFR therapy, we examined the pattern of composite tumor kinase activities governed by EGFR-mediated signaling that might be implicated in development of metastatic disease. Patients and Methods Point mutations in KRAS, BRAF, and PIK3CA and ERBB2 amplification were determined in primary tumors from 63 patients with locally advanced rectal cancer scheduled for radical treatment. Using peptide arrays with tyrosine kinase substrates, ex vivo phosphopeptide profiles were generated from the same baseline tumor samples and correlated to metastasis-free survival. Results Unsupervised clustering analysis of the resulting phosphorylation of 102 array substrates defined two tumor classes, both consisting of cases with and without KRAS/BRAF mutations. The smaller cluster group of patients, with tumors generating high ex vivo phosphorylation of phosphatidylinositol-3-kinase-related substrates, had a particularly aggressive disease course, with almost a half of patients developing metastatic disease within one year of follow-up. Conclusion High phosphatidylinositol-3-kinase-mediated signaling activity of the primary tumor, rather than KRAS/BRAF mutation status, was identified as a hallmark of poor metastasis-free survival in patients with locally advanced rectal cancer undergoing radical treatment of the pelvic cavity.

Individual tumor volume responses to short-course oxaliplatin-containing induction chemotherapy in locally advanced rectal cancer – Targeting the tumor for radiation sensitivity?

BACKGROUND Neoadjuvant treatment of locally advanced rectal cancer (LARC) involves chemoradiotherapy (CRT), which may cause significant toxicity, and the potential role and sequential placement of neoadjuvant chemotherapy (NACT) relative to CRT is under debate. PATIENTS AND METHODS In a non-randomized study of 72 LARC patients, short-course oxaliplatin-containing NACT was administered prior to CRT. Tumor volumes were calculated from magnetic resonance images before and after NACT, and four weeks after CRT, and associations between tumor volume responses and outcome were analyzed. Additionally, the impact of oxaliplatin exposure on radiosensitivity was examined in colorectal carcinoma cell lines. RESULTS All tumors except one responded to NACT, with better responses in T3 than T4 cases, and 69/72 patients obtained additional tumor volume reduction after subsequent CRT. However, no associations were found between tumor volume reduction and long-term outcome. Of note, oxaliplatin-resistant cells were significantly more radiosensitive than the oxaliplatin-sensitive counterparts. CONCLUSIONS Oxaliplatin-containing NACT led to substantial tumor volume reduction with particularly good responses in T3 cases. NACT did not impede subsequent CRT response, and experimental results rather suggested enhanced radiosensitivity in oxaliplatin-exposed cells, encouraging studies to explore the administration of NACT prior to CRT. Data are still lacking to support omitting radiation in LARC management.

Blood pressure control is hard to achieve in patients with chronic renal failure: Results from a survey of renal units in Norway

Objective To assess the use of antihypertensive drugs and blood pressure (BP) levels in relation to current guidelines for BP control in patients with chronic renal failure (CRF). Material and methods A cross-sectional survey was carried out in six renal outpatient clinics in Oslo and the surrounding area. The hospital records of all renal patients not yet in need of renal replacement therapy and with serum creatinine ≥200 μmol/l who attended consultations with nephrologists regularly (at least every third month) were reviewed. Results Of the 351 patients, 97% had hypertension. The majority of patients (96%) were receiving antihypertensive therapy. The average number of antihypertensive drugs being taken was 2.7±1.3 (median 3), but it varied with the cause of CRF. The drugs most frequently prescribed as monotherapy were angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists, which were used by 32%; 51% of patients were using three or more antihypertensive drugs. Loop diuretics were prescribed as monotherapy in 25% of cases and in combination with two or more other drugs in 87%. Age and serum creatinine levels influenced the choice of antihypertensive therapy. The target BP of <130/80 mmHg was obtained in 13% of patients, and lack of optimal BP control was mainly due to systolic hypertension. A total of 38% of patients had a BP of <140/90 mmHg, while 58% failed to achieve a systolic BP of <140 mmHg. Conclusion Optimal blood pressure control is hard to achieve in patients with CRF, even with specialist care and the use of multiple antihypertensive drugs.

Abstract B29: High tumor PI3K signaling in resistance to chemoradiotherapy and metastatic dissemination of locally advanced rectal cancer

Hypoxia is an important hallmark of the tumor microenvironment and a main mechanism in tumor resistance to cytotoxic therapy, local recurrence, and metastatic progression. Locally advanced rectal cancer (LARC) comprises heterogeneous tumors with predominant hypoxic components, growing with locally advanced disease manifestations within the pelvic cavity. Despite the introduction of multimodal therapy, primarily the combination of surgery and radiation that frequently results in local control, a substantial number of patients will experience metastatic dissemination. A rational integration of molecularly targeted therapies in combined-modality regimens might cause both improvement of local control in remaining poor-responding patients and reduction in metastasis risk. This strategy, however, will require a clear definition of functional biomarkers for risk assessment and treatment stratification, and technologies comprising the resultant condition of interacting tumor signaling effects may be particularly advantageous. Within this frame of reference, kinase substrate array technologies are tools for global profiling of kinase activities in tissue samples without prior knowledge of which signaling pathways are activated, theoretically portraying the state of composite information flow through signaling cascades. The present work summarizes our experiences from a prospective study of LARC patients given neoadjuvant chemotherapy and radiation followed by surgery and no further treatment (NCT00278694). Using kinase substrate arrays to analyze study patients9 tumor biopsies sampled at the time of diagnosis, we hypothesized that the approach might enable identification of potentially actionable therapy targets implicated in hypoxic tumor signaling. At first, tumor phosphorylation of array substrates was correlated to histologic tumor response to the neoadjuvant treatment. Essentially, patients with poor response had significantly elevated tumor kinase activity, representing signaling mediated by VEGFR, EGFR, and PI3K, compared to good-responding patients. Next, it appeared that phosphorylation of array substrates representing PDGFR was strongly inhibited by the addition of the anti-angiogenic agent sunitinib to tumor samples from patients without detectable tumor cells in the bone marrow at the time of diagnosis. The recognition that PDGFR-mediated signaling is central for maturation of pericytes during angiogenesis makes it tempting to speculate that intact pericytic signaling of the tumor vasculature is associated with low likelihood of early systemic tumor dissemination in LARC. Finally, unsupervised clustering analysis of the array phosphosubstrate data separated the tumor samples into two phenotypic populations. The smaller of the patient groups, displaying high tumor activities within the PI3K signaling network, showed a particularly aggressive disease course as almost a half of cases had developed metastatic disease by less than one year of follow-up, suggesting a functional biomarker for rapid failure of metastatic disease control following radical treatment of the pelvic cavity. Given our findings that PI3K may be a key signaling orchestrator both of poor local tumor response to neoadjuvant treatment and importantly, of metastatic development, therapeutic targeting of components of the PI3K complex might be rational to integrate into combined-modality treatment regimens in LARC with hypoxic tumor features. Citation Format: Anne Hansen Ree, Kjersti Flatmark, Marie Gron Saelen, Sigurd Folkvord, Svein Dueland, Jurgen Geisler, Kathrine Roe. High tumor PI3K signaling in resistance to chemoradiotherapy and metastatic dissemination of locally advanced rectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B29.

Abstract 3780: On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells

Tumor hypoxia, causing metabolic changes towards enhanced glycolysis and lactic acid production, is a common microenvironmental feature of solid tumors, and is recognized as a main mechanism both of therapeutic resistance and metastasis. The hypoxia-inducible factor-1α conducts transcription of a number of genes involved in adaption to the hypoxic condition. Among these is the tumor-specific carbonic anhydrase IX (CAIX), a membrane-bound protein that regulates extracellular (pHe) and intracellular (pHi) pH by catalyzing the reversible hydration of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 - ) and protons, enabling tumor cell survival in the acidic microenvironment. To improve therapeutic outcome and reduce metastatic risk, targeted inhibition of CAIX is an attractive strategy. Here we report on effects of a CAIX inhibitor in a panel of colorectal carcinoma (CRC) cell lines, giving specific emphasis to the microenvironmental conditions. Five CRC cell lines were incubated for 24 hours under normoxic (21% O 2 ) or hypoxic (0.2% O 2 ) condition, either in the presence (22 mM HCO 3 - , 5% CO 2 ) or absence (0 mM HCO 3 - , 0% CO 2 ) of HCO 3 - in the culture medium. The cells were treated with 4-(3′-(3”,5”-dimethylphenyl)ureido)phenyl sulfamate (termed S4), considered to be a CAIX-specific inhibitor, and clonogenicity, pHe, and pHi (measured as cells labeled with a pH-sensitive dye) were assessed. In CO 2 /HCO 3 - -buffered medium under normoxia, differential inhibition of clonogenicity following S4 treatment was observed among the five cell lines, correlating with individual CAIX levels: strong suppression in cell lines with high CAIX expression, weaker S4 effect in cell lines with low expression, and no effect in the CAIX-negative cell line. When incubating with this medium under hypoxia, despite significant induction of CAIX, no additional effect of S4 on clonogenicity was observed. Of note, when changing the cell culture medium to CO 2 /HCO 3 - -free condition, S4 strongly inhibited clonogenicity of hypoxic HT29 cells (high CAIX expression) but not of the normoxic counterpart, while HCT116 cells (low CAIX expression) showed no clonogenic response to S4 with either oxygenation status. In all of the five cell lines, hypoxia caused significant acidification (decrease in pHe) of culture media. In parallel with the clonogenic response, S4 treatment significantly counteracted the hypoxia-induced pHe decline in HT29 cultures incubated in the CO 2 /HCO 3 - -free condition. pHi was not altered with any of the tested conditions. This set of data demonstrated that the CO 2 /HCO 3 - buffering system determined CRC cell response to targeted CAIX inhibition. To enable reliable assessment of experimental S4 effects, strict control of the microenvironmental conditions was required. We believe this information may be of note for future evaluation of this specific therapeutic approach. Citation Format: Helga Helseth Hektoen, Kjersti Flatmark, Kirsti Solberg Landsverk, Marie Gron Saelen, Kathrine Roe, Anne Hansen Ree. On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2014-3780

Abstract C63: The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity.

In modern radiation oncology, new insights into molecular radiobiology provide an opportunity for the rational integration of molecularly targeted therapeutics to optimize clinical radiation effects. One example is the use of histone deacetylase (HDAC) inhibitors as potentially radiosensitizing drugs. Conveyed by histone acetylation, HDAC inhibition causes perturbations in gene regulation implicated in cell cycle progression, DNA damage signaling and repair, and apoptosis. Following the demonstration that HDAC inhibitors enhanced radiation-induced clonogenic suppression in human colorectal carcinoma cell lines and xenograft models [1-3], the PRAVO study was conducted [4-5]. This trial, undertaken in patients treated with pelvic palliative radiotherapy (30 Gy in 3-Gy daily fractions) combined with the HDAC inhibitor vorinostat (administered once daily, three hours before radiation) for advanced gastrointestinal malignancy, was the first to report on the use of an HDAC inhibitor in clinical radiotherapy. It was designed to demonstrate that vorinostat reached the specific target (detection of tumor histone acetylation), the applicability of non-invasive tumor response assessment (using functional imaging), and importantly, that this combined-modality therapy was safe and tolerable. In the present report, potential biomarkers of vorinostat radiosensitizing action, not simultaneously manifesting molecular perturbations elicited by the radiation itself, were explored by gene expression array analysis of the PRAVO study patients’ peripheral blood mononuclear cells (PBMC), sampled at baseline (T0) and on-treatment two and 24 hours (T2 and T24) after the patients had received vorinostat. This strategy revealed 1,600 array probes that were common for the comparisons T2 versus T0 and T24 versus T2 across all of the patients, and furthermore, that no significantly differential expression was observed between the T0 and T24 groups. Functional annotation analysis of the array data showed that a significant number of the identified genes were implicated in biological processes and pathways comprising gene regulation (transcription, RNA processing), cell cycle progression (including p53 signaling, commonly involved in the DNA damage response), and chromatin biology. Of five genes that were selected both for verification of patients’ PBMC expression and for validation of vorinostat-regulated expression in human colorectal carcinoma xenograft models, transient repression of MYC was consistently observed in all conditions. In conclusion, within the design of the PRAVO study, all of the identified genes showed rapid and transient induction or repression and therefore, in principle, fulfilled the requirement of being pharmacodynamic biomarkers of vorinostat activity in fractionated radiotherapy, possibly underscoring the regulatory role of myc in this therapeutic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C63. Citation Format: Anne Hansen Ree, Marie G. Saelen, Erta Kalanxhi, Ingrid H. G. Ostensen, Kristina Schee, Kathrine Roe, Torveig W. Abrahamsen, Svein Dueland, Kjersti Flatmark. The Pelvic Radiation and Vorinostat (PRAVO) phase 1 study identifying MYC repression as biomarker of histone deacetylase inhibitor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C63.