Mutant PIK3CA controls DUSP1-dependent ERK 1/2 activity to confer response to AKT target therapy.

BACKGROUND: Alterations in the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway are frequent in urothelial bladder cancer (BLCA) and thus provide a potential target for novel therapeutic strategies. We investigated the efficacy of the AKT inhibitor MK-2206 in BLCA and the molecular determinants that predict therapy response. METHODS: Biochemical and functional effects of the AKT inhibitor MK-2206 were analysed on a panel of 11 BLCA cell lines possessing different genetic alterations. Cell viability (CellTiter-Blue, cell counts), apoptosis (caspase 3/7 activity) and cell cycle progression (EdU incorporation) were analysed to determine effects on cell growth and proliferation. cDNA or siRNA transfections were used to manipulate the expression of specific proteins such as wild-type or mutant PIK3CA, DUSP1 or CREB. For in vivo analysis, the chicken chorioallantoic membrane model was utilised and tumours were characterised by weight and biochemically for the expression of Ki-67 and AKT phosphorylation. RESULTS: Treatment with MK-2206 suppressed AKT and S6K1 but not 4E-BP1 phosphorylation in all cell lines. Functionally, only cell lines bearing mutations in the hotspot helical domain of PIK3CA were sensitive to the drug, independent of other genetic alterations in the PI3K or MAPK signalling pathway. Following MK-2206 treatment, the presence of mutant PIK3CA resulted in an increase in DUSP1 expression that induced a decrease in ERK 1/2 phosphorylation. Manipulating the expression of mutant or wild-type PIK3CA or DUSP1 confirmed that this mechanism is responsible for the induction of apoptosis and the inhibition of tumour proliferation in vitro and in vivo, to sensitise cells to AKT target therapy.Conclusion or interpretation:PIK3CA mutations confer sensitivity to AKT target therapy in BLCA by regulating DUSP1 expression and subsequent ERK1/2 dephosphorylation and can potentially serve as a stratifying biomarker for treatment.

Localization of topoisomerase II in mitotic chromosomes.

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.

Multiple replication origins are used during Drosophila chorion gene amplification.

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.

Topoisomerase II: A specific marker for cell proliferation.

We have used an antibody probe to measure the levels of topoisomerase II in several transformed and developmentally regulated normal cell types. Transformed cells contain roughly 1 X 10(6) copies of the enzyme. During erythropoiesis in chicken embryos the enzyme level drops from 7.8 X 10(4) copies per erythroblast to less than 300 copies per erythrocyte concomitant with the cessation of mitosis in the blood. Cultured myoblasts also lose topoisomerase II upon fusion into nonproliferating myotubes. When peripheral blood lymphocytes (which lack detectable topoisomerase II) commence proliferation, they express topoisomerase II de novo. Appearance of the enzyme exactly parallels the onset of DNA replication. These results suggest that topoisomerase II is not required for transcription in higher eukaryotes, but that it may function during DNA replication. Furthermore, topoisomerase II is a sensitive and specific marker for proliferating cells.

The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.

We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.

The kinesin-like protein KLP61F is essential for mitosis in Drosophila.

We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.

[Molecular tumor board prostate cancer]. / Molekulares Tumorboard Prostatakarzinom.

In modern oncology, molecular tumor boards are the interface between the public healthcare system and clinical research institutions. An interdisciplinary team of medical and scientific experts assesses if extensive molecular testing for tumor profiling is appropriate and discusses therapeutic options for patients with newly diagnosed treatable alterations. In the field of metastatic prostate cancer, patients especially with a strong family history, young age of diagnosis and those who have exhausted standard treatments may benefit from molecular profiling. Expression of the androgen receptor splice variant 7 (AR-V7) predicts nonresponse to next-generation AR-directed therapy like abiraterone or enzalutamide. Different blood tests for AR-V7 detection are now commercially available. Mutations in the DNA repair pathway are another frequent event in metastatic prostate cancer. Homologous recombination defects sensitize cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors. In the TOPARP-A trial, the PARP inhibitor olaparib led to high response rates (88%) in patients with mutated DNA repair genes. Furthermore, patients with DNA mismatch repair deficiency and/or microsatellite instability seem to benefit from PD-1 inhibitors, particularly pembrolizumab. At this time neither PARP inhibitors nor PD-1 inhibitors are approved for metastatic prostate cancer treatment in Germany. Therefore, a recommendation of a molecular tumor board for biomarker-matched off-label use of approved drugs across entity barriers will support coverage by health insurance.

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis.

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

The Drosophila RAD21 cohesin persists at the centromere region in mitosis.

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.

Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.

[PSMA-targeted radioligand therapy in prostate cancer]. / Radionuklidtherapie des Prostatakarzinoms mittels PSMA-Lutetium.

Radioligand therapy (RLT) directed against prostate-specific membrane antigen (PSMA) enables tumor-specific treatment directed against PSMA-overexpressing prostate cancer cells. Several PSMA ligands such as PSMA-617 or PSMA-I&T have been developed that can be labeled with ß­radiating lutetium-177. These are currently applied in compassionate use programs to treat metastatic castration-resistant prostate cancer (mCRPC). PSMA-directed RLT is currently being offered in several nuclear medicine departments throughout Germany. Several retrospective case series demonstrate its activity with a prostate-specific antigen (PSA) decrease >50% in 30-60% of mCRPC patients. The toxicity seems to be low. Hematologic grade 4 toxicity has not been observed and grade 3 toxicities rarely occur. The main nonhematologic adverse events are intermittent dry mouth because of unspecific PSMA expression in the salivary glands as well as fatigue and nausea. Currently there are no prospective studies available for evaluation of PSMA-targeted RLT and a survival benefit over approved standard therapies such as abiraterone, enzalutamide, radium-223-dichloride, docetaxel or cabazitaxel has not been shown. PSMA-targeted RLT should therefore currently only be offered after critical evaluation in patients who exhausted the approved standard therapies.

Circulating tumor cells versus objective response assessment predicting survival in metastatic castration-resistant prostate cancer patients treated with docetaxel chemotherapy.

PURPOSE: Circulating tumor cell (CTC) counts might display a superior prognostic value for overall survival (OS) compared to objective response criteria (OR) in metastatic castration-resistant prostate cancer (mCRPC) patients. METHODS: CTCs were detected using the CellSearch™ System out of 122 samples during docetaxel chemotherapy (75 mg/m(2)) at baseline (q0) and after 1 (q1), 4 (q4) and 10 (q10) cycles, in mCRPC patients (n = 33). OR was evaluated by morphologic RECIST and clinical criteria after 4 (q4) and 10 (q10) cycles. RESULTS: For OS, analyses revealed a significant prognostic value for categorical (<5 vs. ≥5) CTC counts (q0, p = 0.005; q1, p = 0.001; q4, p < 0.001; q10, p = 0.002), RECIST (q4, p < 0.001; q10, p = 0.02) and clinical criteria (q4, p < 0.001; q10, p = 0.02). Concordance of CTC counts with OR revealed a sensitivity of 83.3-87.5 % and a specificity of 68.0-76.5 % with complementary discriminatory power for OS. Comparing CTC counts with concomitant OR at q4 in multivariate analyses, an independent prognostic value for OS was found for CTC counts (HR 3.3; p = 0.02) similar to clinical (HR 4.9; p = 0.02) and radiologic response (HR 3.4; p = 0.051). Comparing the predictive value for death, early post-treatment CTC counts at q1 demonstrated significant accuracy with an area under the curve of 79.5 % (p = 0.004) similar to CTC counts at q4 (76.7 %; p = 0.009). Radiologic and clinical response at q4 displayed accuracy similar to early CTC counts at q1 (72.2 %; p = 0.03 and 75.0 %; p = 0.02) despite low sensitivities. CONCLUSIONS: CTC counts appear to be an earlier and more sensitive predictor for survival and treatment response than current OR approaches and may provide complementary information toward individualized treatment strategies.

[Molecular lymph node staging in prostate and bladder cancer]. / Molekulares Lymphknotenstaging beim Prostata- und Harnblasenkarzinom.

A positive lymph node status is a major prognostic factor for tumor recurrence and mortality following radical prostatectomy in prostate cancer patients or radical cystectomy in bladder cancer patients. However, despite having histopathologic negative lymph nodes, a substantial proportion of patient suffers from tumor recurrence within a few years after the operation. Tumor recurrence in node-negative patients may result from hematogeneous or lymphatic metastatic spread which remains undetected by standard clinical or histopathologic examinations. Molecular lymph node analysis is a potential method for detection of lymph node metastases with higher sensitivity and for prognostic risk stratification of patients with histopathologic negative lymph nodes.

[Detection of circulating tumor cells from peripheral blood in prostate cancer]. / Detektion zirkulierender Tumorzellen im peripheren Blut beim Prostatakarzinom.

Circulating tumor cells (CTCs) can be detected with sensitive immunocytological and molecular methods and can potentially cause distant metastases. Consecutively the prognostic significance of CTC-counts for survival was demonstrated in metastatic prostate cancer (mPC) revealing CTCs as reliable surrogate marker during therapy. Comparatively the prognostic value of a CTC-threshold with < 5 vs. ≥ 5 CTCs was superior to the commonly used PSA-decrement algorithms. In contrast despite evidence of CTCs in localized PC, their clinical value in this stage is currently precarious. Furthermore, CTCs may serve as predictive markers with the ability to predict treatment sensitivity or resistance, since they may represent the heterogeneous molecular signature of primary as well as metastatic cancer lesions. Thus, the isolation of CTCs may serve not only as a prognostic tool but moreover as a liquid biopsy and a window towards personalized treatment. This review discusses the clinical impact of CTCs in the different stages of PC.

[Chemotherapy for bladder cancer: 2012 Update. From AUO ("Arbeitsgemeinschaft Urologische Onkologie") and IABC ("Interdisziplinäre Arbeitsgruppe BlasenCarcinom")]. / Chemotherapie beim Harnblasenkarzinom: Update 2012. Aus AUO (Arbeitsgemeinschaft Urologische Onkologie) und IABC (Interdisziplinäre Arbeitsgruppe BlasenCarcinom).

This article summarizes contemporary standards in systemic therapy for urothelial carcinoma as well as updated results of peer-reviewed publications and international bladder cancer meetings in 2011. Both neoadjuvant and adjuvant trials for locally advanced carcinoma and data on systemic chemotherapy for metastatic bladder cancer are discussed.

[Compassionate use of abiraterone and cabazitaxel: first experiences in docetaxel-pretreated castration-resistant prostate cancer patients]. / Abiraterone- und Cabazitaxel-Härtefallprogramm : Erste klinische Erfahrungen bei Docetaxel-vorbehandelten kastrationsresistenten Prostatakarzinompatienten.

OBJECTIVE: First clinical experiences with abiraterone and cabazitaxel for the treatment of metastatic castration-resistant prostate cancer patients following docetaxel chemotherapy are reported. PATIENTS AND METHODS: We describe PSA response rates and disease control rates determined by imaging studies at 3 months as well as side effects in the daily routine. All patients were treated within the"compassionate use" programs of cabazitaxel and abiraterone or treated according to their inclusion and exclusion criteria at the "Technische Universität München". RESULTS: Of 54 patients, 15 were treated with cabazitaxel and 39 with abiraterone. In patients treated with cabazitaxel, after 3 months of therapy the PSA reduction rate > 50% was 46.2%, the PSA progression rate was 15.4%, and the disease control rate was 83.3%. Main grade 3/4 hematotoxicities were neutropenia (40%) and anemia (20%). Febrile neutropenia was observed in 2 of 15 (13.3%) patients. Main non-hematological grade 3/4 toxicities were diarrhea (13.3%) and polyneuropathy (13.3%). In patients treated with abiraterone, after 3 months of therapy the PSA reduction rate >50% was 35.1%, the PSA progression rate was 46.0%, and the disease control rate was 47.1%. Main grade 3/4 hematotoxicities were anemia (5.1%) and thrombocytopenia (5.1%). Main non-hematological toxicities were fatigue (20.5%), sweating (17.9%), and constipation (10.3%). CONCLUSION: Utilization of cabazitaxel and abiraterone in the daily routine show response rates comparable to their approval studies with acceptable side effects.

[Update on chemotherapy for bladder cancer. Update 2010]. / Chemotherapie beim Harnblasenkarzinom. Update 2010.

This review summarizes results of neoadjuvant and adjuvant trials for locally advanced bladder cancer as well as data on systemic chemotherapy for metastatic urothelial cancer. Increasing attention has been drawn to targeted cancer therapies in the treatment of urothelial cancer in recent years. In future, molecular profiling will play a major role as a means to tailor individual therapy.