Episodic Src activation in uveal melanoma revealed by kinase activity profiling

Background:

The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM.

Methods:

We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays.

Results:

We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced.

Conclusion:

We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.     

The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

Background:

Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines.

Methods:

Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines.

Results:

All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations.

Conclusion:

These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.     

A phase I trial of vertical inhibition of IGF signalling using cixutumumab,an anti-IGF-1R antibody,and selumetinib,an MEK 1/2 inhibitor,in advanced solid tumours

Background:

We completed a phase I clinical trial to test the safety and toxicity of combined treatment with cixutumumab (anti-IGF-1R antibody) and selumetinib (MEK 1/2 inhibitor).

Methods:

Patients with advanced solid tumours, refractory to standard therapy received selumetinib hydrogen sulphate capsules orally twice daily, and cixutumumab intravenously on days 1 and 15 of each 28-day cycle. The study used a 3+3 design, with a dose-finding cohort followed by an expansion cohort at the maximally tolerated dose that included pharmacokinetic and pharmacodynamic correlative studies.

Results:

Thirty patients were enrolled, with 16 in the dose-finding cohort and 14 in the expansion cohort. Grade 3 or greater toxicities included nausea and vomiting, anaemia, CVA, hypertension, hyperglycaemia, and ophthalmic symptoms. The maximally tolerated combination dose was 50 mg twice daily of selumetinib and 12 mg kg⁻¹ every 2 weeks of cixutumumab. Two patients achieved a partial response (one unconfirmed), including a patient with BRAF wild-type thyroid carcinoma, and a patient with squamous cell carcinoma of the tongue, and six patients achieved time to progression of >6 months, including patients with thyroid carcinoma, colorectal carcinoma, and basal cell carcinoma. Comparison of pre- and on-treatment biopsies showed significant suppression of pERK and pS6 activity with treatment.

Conclusions:

Our study of anti-IGF-1R antibody cixutumumab and MEK 1/2 inhibitor selumetinib showed that the combination is safe and well-tolerated at these doses, with preliminary evidence of clinical benefit and pharmacodynamic evidence of target inhibition.     

Correlation of BRAF and NRAS mutation status with outcome,site of distant metastasis and response to chemotherapy in metastatic melanoma

Background:

The prognostic significance of BRAF and NRAS mutations in metastatic melanoma patients remains uncertain, with several studies reporting conflicting results, often biased by the inclusion of patients treated with BRAF and MEK (MAPK) inhibitors. We therefore interrogated a historical cohort of patients free of the confounding influence of MAPK inhibitor therapy.

Methods:

Patients with available archival tissue first diagnosed with metastatic melanoma between 2002 and 2006 were analysed. Mutational analysis was performed using the OncoCarta Panel. Patient characteristics, treatment outcome and survival were correlated with BRAF/NRAS mutation status.

Results:

In 193 patients, 92 (48%) melanomas were BRAF-mutant, 39 (20%) were NRAS-mutant and 62 (32%) were wild-type for BRAF/NRAS mutations (wt). There was no difference in response to chemotherapy based on mutation status (35–37%). The distant disease-free interval (DDFI) was significantly shorter in patients with wt melanoma (27.9 months vs 35.1 for BRAF and 49.1 for NRAS) although this was not significant in multivariate analysis. Survival from stage IV melanoma diagnosis was not significantly different based on mutation status. The DDFI was significantly shorter in patients with BRAF^V600K/R versus BRAF^V600E melanoma in univariate and multivariate analyses.

Conclusions:

BRAF and NRAS mutation status does not influence survival in metastatic melanoma.     

Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase A activation in human lung and colorectal cancer cells

Background:

MEK is activated in ∼40% colorectal cancer (CRC) and 20–30% non-small cell lung cancer (NSCLC). Selumetinib is a selective inhibitor of MEK1/2, which is currently in clinical development.

Methods:

We evaluated the effects of selumetinib in vitro and in vivo in CRC and NSCLC cell lines to identify cancer cell characteristics correlating with sensitivity to MEK inhibition.

Results:

Five NSCLC and six CRC cell lines were treated with selumetinib and classified according to the median inhibitory concentration (IC₅₀) values as sensitive (⩽1 μℳ) or resistant (>1 μℳ). In selumetinib-sensitive cancer cell lines, selumetinib treatment induced G1 cell-cycle arrest and apoptosis and suppression of tumour growth as xenografts in immunodeficient mice. Evaluation of intracellular effector proteins and analysis of gene mutations showed no correlation with selumetinib sensitivity. Microarray gene expression profiles revealed that the activation of cAMP-dependent protein kinase A (PKA) was associated with MEK inhibitor resistance. Combined targeting of both MEK and PKA resulted in cancer cell growth inhibition of MEK inhibitor-resistant cancer cell lines in vitro and in vivo.

Conclusion:

This study provides molecular insights to explain resistance to an MEK inhibitor in human cancer cell lines.     

Optimising the combination dosing strategy of abemaciclib and vemurafenib in BRAF-mutated melanoma xenograft tumours

Background:

Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation.

Methods:

A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival.

Results:

The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression.

Conclusions:

The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.     

Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

Background:

Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood.

Methods:

Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation.

Results:

While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex.

Conclusion:

Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis.     

α-TEA cooperates with MEK or mTOR inhibitors to induce apoptosis via targeting IRS/PI3K pathways

Background:

α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers.

Methods:

Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques.

Results:

α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them.

Conclusions:

Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.     

ERK5 signalling in prostate cancer promotes an invasive phenotype

Background:

Aberrant mitogen/extracellular signal-regulated kinase 5 (MEK5)–extracellular signal-regulated protein kinase 5 (ERK5)-mediated signalling has been implicated in a number of tumour types including prostate cancer (PCa). The molecular basis of ERK5-driven carcinogenesis and its clinical relevance remain to be fully characterised.

Methods:

Modulation of ERK5 expression or function in human PCa PC3 and PC3–ERK5 (stably transfected with ERK5) cells was performed using siRNA-mediated knockdown or the MEK inhibitor PD18435 respectively. In vitro significance of ERK5 signalling was assessed by assays for proliferation, motility, invasion and invadopodia. Expression of matrix metalloproteinases/tissue inhibitors of metalloproteases was determined by Q-RT–PCR. Extracellular signal-regulated protein kinase 5 expression in primary and metastatic PCa was examined using immunohistochemistry.

Results:

Reduction of ERK5 expression or signalling significantly inhibited the motility and invasive capability of PC3 cells. Extracellular signal-regulated protein kinase 5-mediated signalling significantly promoted formation of in vivo metastasis in an orthotopic PCa model (P<0.05). Invadopodia formation was also enhanced by forced ERK5 expression in PC3 cells. Furthermore, in metastatic PCa, nuclear ERK5 immunoreactivity was significantly upregulated when compared with benign prostatic hyperplasia and primary PCa (P=0.013 and P<0.0001, respectively).

Conclusion:

Our in vitro, in vivo and clinical data support an important role for the MEK5–ERK5 signalling pathway in invasive PCa, which represents a potential target for therapy in primary and metastatic PCa.     

Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression

Background:

Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423–7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.

Methods:

We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAF^V600E mutation, as well as by cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.

Results:

Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells. Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.

Conclusion:

Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.     

KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition

Background:

To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines.

Methods:

A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry.

Results:

We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines.

Conclusions:

Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.     

Impact of mutational status on survival in low-grade serous carcinoma of the ovary or peritoneum

Background:

Low-grade serous carcinoma of the ovary or peritoneum is a distinct, well- recognized histologic subtype characterized by young age at diagnosis, relative chemoresistance, and prolonged overall survival. Common mutations reported to be found within this subtype include KRAS and BRAF.

Methods:

Using clinical information of patients from our IRB-approved registry and tissue from a subset of these patients, we performed mutational analysis for KRAS and BRAF using the direct Sanger sequencing technique and correlated findings with the clinical outcome, overall survival (OS).

Results:

In 79 cases, patients with KRAS or BRAF mutations (n=21) had a significantly better OS than those with wild-type KRAS or BRAF (n=58) (106.7 months (95% CI, 50.6, 162.9) vs 66.8 months (95% CI, 43.6, 90.0)), respectively (P=0.018).

Conclusions:

Mutational status appears to be a potential prognostic factor in low-grade serous carcinoma of the ovary or peritoneum.     

The MEK1/2 inhibitor, selumetinib (AZD6244; ARRY-142886), enhances anti-tumour efficacy when combined with conventional chemotherapeutic agents in human tumour xenograft models

Background:

The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy.

Methods:

The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations.

Results:

Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib.

Conclusion:

The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.     

Role of endoplasmic reticulum stress induction by the plant toxin,persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells

Background:

Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved.

Methods:

The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference.

Results:

Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells.

Conclusion:

The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.     

Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion

Background:

Focal adhesion kinase (FAK) is overexpressed in a variety of cancers, such as breast, colon, prostate, ovary, and lung cancers. However, the mechanism by which extracellular matrix fibronectin stimulates lung cancer cell migration and invasion through FAK remains to be investigated.

Methods:

The signalling pathways in fibronectin-mediated lung cancer cell migration and invasion were examined using western blotting. The metastasis function was detected by wound healing, migration and invasion assays. Further, RNA interference and kinase inhibitors were also used to study the downstream signals.

Results:

In this study, we examined the FAK signalling pathways in relation to calpain-2 and RhoA in fibronectin-mediated lung cancer cell migration and invasion. We found that A549 lung epithelial cells stimulated by fibronectin showed increased phosphorylation of FAK and its downstream targets, Src, ERK1/2, phosphatidylinositol 3′-kinase (PI3K), and Akt. Consistent with this observation, depletion of FAK by siRNA resulted in the inhibition of Src, ERK1/2, PI3K, and Akt activity. In addition, the Src inhibitor, PP2, blocked the phosphorylation of FAK, ERK1/2, PI3K, and Akt. Conversely, inhibition of MEK1/2 using PD98059 reduced the expression of matrix metalloproteinase-9 (MMP9) and calpain-2. The PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, further blocked the expression of MMP9 and RhoA. Inhibition of both MEK1/2 and PI3K caused reduced cell migration and invasion.

Conclusion:

Our data suggest that fibronectin-mediated activation of FAK that leads to lung cancer metastasis could occur through ERK or PI3K/Akt regulation of MMP9/calpain-2 or MMP9/RhoA activity, respectively.     

Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability

Background:

Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood.

Methods:

In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity.

Results:

We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling.

Conclusions:

Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance.     

Notch1 regulates the functional contribution of RhoC to cervical carcinoma progression

Background:

The role of Notch signalling in human epithelial cancers is of immense interest. In this study, we examine the interplay between Notch signalling and RhoC, a well-established molecular factor in metastasis. By linking the function of Notch and RhoC, we further strengthen the notion that there is a pro-oncogenic role of Notch signalling in human cervical cancers.

Methods:

RhoC protein expression in cervical carcinoma cell lines was assessed by western blotting. Using CaSki and SiHa cells (cervical carcinoma cells lines), we show that RhoC contributes to wound healing, invasion and migration, anoikis resistance, colony formation, in vitro tube formation and tumour formation. Immunohistochemical studies were carried out to assess the co-expression of RhoC, pAkt and Notch1 in clinical sections.

Results:

An assessment of the changes associated with epithelial-to-mesenchymal transition (EMT) shows that both Notch1 and RhoC have similar phenotypic contribution to EMT. Rho activity assessment on Notch1 inhibition with DAPT shows decreased RhoC activity. We further show that constitutively active RhoC rescues the phenotypic effect of Notch1 inactivation, and a comparison of Notch1 with RhoC expression shows an overlap between the two proteins in the same areas of the tissue.

Conclusion:

This study has provided evidence to suggest that RhoC is an effector of Notch1 in cervical carcinoma.     

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:

Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.

Methods:

Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.

Results:

Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.

Conclusion:

Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.     

Rare BRAF mutations in melanoma patients: implications for molecular testing in clinical practice

Background:

The detection of V600E BRAF mutation in melanoma is fundamental since here BRAF inhibitors represent an effective treatment. Non-V600E BRAF mutations that may also respond are not detected by certain screening methods. Thus, knowledge about detection of these mutations is needed.

Methods:

A total of 276 tumour samples from 174 melanoma patients were investigated for BRAF mutations by pyrosequencing. Rare mutations were confirmed by capillary sequencing and compared with findings from COBAS test and immunohistochemistry using a novel BRAF antibody. Melanoma type, localisation, and survival were summarised.

Results:

BRAF mutations were found in 43% of patients (124 tumours in 75 patients). Among those, 14 patients (18.7%) exhibited rare mutations. The V600EK601del and V600DK601del mutations have never been described before in melanoma. Furthermore, V600K, V600E2, and V600D, V600G, V600R, and L597S mutations were detected. Mutations were not detected by COBAS test in 7 out of these 14 patients and immunohistochemistry only reliably detected patients with the V600E2 and V600EK601del mutation.

Conclusion:

Accurate diagnosis of rare BRAF mutations is crucial. We show that pyrosequencing is accurate, highly sensitive, reliable, and time saving to detect rare BRAF mutations. Missing these rare variant mutations would exclude a subset of patients from available effective BRAF-targeting therapy.