Mutant p53: a novel target for the treatment of patients with triple-negative breast cancer?

The identification and validation of a targeted therapy for patients with triple-negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti-cancer activity of PRIMA-1 and PRIMA-1MET (APR-246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild-type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA-1 and PRIMA-1MET was found to be cell-line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA-1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=-0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA-1MET induced apoptosis and inhibited migration in a p53 mutant-dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA-1MET is a potential new approach for treating p53-mutated breast cancer, including the subgroup with triple-negative (TN) disease.

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells.

BACKGROUND: Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC. METHODS: We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines. RESULTS: D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells. CONCLUSION: Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.

Tumor markers in colorectal cancer, gastric cancer and gastrointestinal stromal cancers: European group on tumor markers 2014 guidelines update.

Biomarkers currently play an important role in the detection and management of patients with several different types of gastrointestinal cancer, especially colorectal, gastric, gastro-oesophageal junction (GOJ) adenocarcinomas and gastrointestinal stromal tumors (GISTs). The aim of this article is to provide updated and evidence-based guidelines for the use of biomarkers in the different gastrointestinal malignancies. Recommended biomarkers for colorectal cancer include an immunochemical-based fecal occult blood test in screening asymptomatic subjects ≥50 years of age for neoplasia, serial CEA levels in postoperative surveillance of stage II and III patients who may be candidates for surgical resection or systemic therapy in the event of distant metastasis occurring, K-RAS mutation status for identifying patients with advanced disease likely to benefit from anti-EGFR therapeutic antibodies and microsatellite instability testing as a first-line screen for subjects with Lynch syndrome. In advanced gastric or GOJ cancers, measurement of HER2 is recommended in selecting patients for treatment with trastuzumab. For patients with suspected GIST, determination of KIT protein should be used as a diagnostic aid, while KIT mutational analysis may be used for treatment planning in patients with diagnosed GISTs.

ADAM-17: a novel therapeutic target for triple negative breast cancer.

BACKGROUND: Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS: Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS: In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION: It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.

The war on cancer: are we winning?

Of all the diseases affecting humankind, cancer is one of the most difficult to treat and cure. One of the main reasons for this difficulty relates to the fact that cancer is not a single disease but consists of hundreds of different types. Furthermore, cancers exhibit considerable genetic complexity with more than 400 different genes implicated in their development. In addition, cancers display major inter- and intratumor heterogeneity. Despite these complexities, several successes have been achieved in recent years. Most of these successes relate to the specific targeting of driver genes involved in cancer development. These successes include imatinib for the treatment of chronic myeloid leukemia, anti-HER2 therapies (trastuzumab, pertuzumab, and lapatinib) to treat breast cancer, anti-EGFR tyrosine kinase inhibitors (gefitinib and erlotinib) to treat non-small cell lung cancer, and anti-BRAF agents (vemurafenib and dabrafenib) to treat melanoma. Although the war on cancer has not yet been won, neither has it been lost. With continued basic and clinical research, cancer is being transformed into a chronic disease in which patients have increased survival rates and better quality of life.

Trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in HER-2-non-amplified breast cancer cell lines.

BACKGROUND: Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD56+natural killer (NK) cells may contribute to the activity of trastuzumab in HER-2-amplified tumours. In this study, we investigated the possibility that trastuzumab might induce ADCC against HER-2-non-amplified breast cancer cells. METHODS: Induction of NK cell-mediated ADCC was examined in trastuzumab-treated HER-2-non-amplified breast cancer cell lines. HER-2 protein levels were also determined in tumour and autologous normal tissue samples from patients with HER-2 negative breast cancer. RESULTS: Trastuzumab induced a significant ADCC response in the HER-2-amplified HCC1954 and SKBR3 cell lines, and in all five of the non-amplified cell lines, which had low levels of detectable HER-2 by western blot (CAL-51, CAMA-1, MCF-7, T47D, and EFM19). Trastuzumab did not induce ADCC in the K562 control cell line or MDA-MB-468, which has very low levels of HER-2 detectable by enzyme-linked immunosorbent assay (ELISA) only. HER-2 protein was detected by ELISA in 14/15 patient tumour samples classified as HER-2-non-amplified. Significantly lower levels of HER-2 were detected in normal autologous tissue compared with tumour samples from the same patients. CONCLUSION: Our results suggest that HER-2-non-amplified breast cancer cells, with low but detectable levels of HER-2 protein, can bind trastuzumab and initiate ADCC.

LIAD-fs scheme for studies of ultrafast laser interactions with gas phase biomolecules.

Laser induced acoustic desorption (LIAD) has been used for the first time to study the parent ion production and fragmentation mechanisms of a biological molecule in an intense femtosecond (fs) laser field. The photoacoustic shock wave generated in the analyte substrate (thin Ta foil) has been simulated using the hydrodynamic HYADES code, and the full LIAD process has been experimentally characterised as a function of the desorption UV-laser pulse parameters. Observed neutral plumes of densities >10(9) cm(-3) which are free from solvent or matrix contamination demonstrate the suitability and potential of the source for studying ultrafast dynamics in the gas phase using fs laser pulses. Results obtained with phenylalanine show that through manipulation of fundamental femtosecond laser parameters (such as pulse length, intensity and wavelength), energy deposition within the molecule can be controlled to allow enhancement of parent ion production or generation of characteristic fragmentation patterns. In particular by reducing the pulse length to a timescale equivalent to the fastest vibrational periods in the molecule, we demonstrate how fragmentation of the molecule can be minimised whilst maintaining a high ionisation efficiency.

Inhibition of IGF1R activity enhances response to trastuzumab in HER-2-positive breast cancer cells.

BACKGROUND: although trastuzumab has improved the prognosis for HER-2-positive breast cancer patients, not all HER-2-positive breast tumours respond to trastuzumab treatment and those that initially respond frequently develop resistance. Insulin-like growth factor-1 receptor (IGF1R) signalling has been previously implicated in trastuzumab resistance. We tested IGF1R inhibition to determine if dual targeting of HER-2 and IGF1R improves response in cell line models of acquired trastuzumab resistance. MATERIALS AND METHODS: HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by enzyme-linked immunosorbent assays in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using both small interfering RNA (siRNA) and the tyrosine kinase inhibitor, NVP-AEW541. RESULTS: IGF1R levels were significantly increased in the trastuzumab-resistant model, SKBR3/Tr, compared with the parental SKBR3 cell line. In both the SKBR3/Tr and BT474/Tr cell lines, inhibition of IGF1R expression with siRNA or inhibition of tyrosine kinase activity by NVP-AEW541 significantly increased response to trastuzumab. The dual targeting approach also improved response in the parental SKBR3 cells but not in the BT474 parental cells. CONCLUSIONS: our results confirm that IGF1R inhibition improves response to trastuzumab in HER-2-positive breast cancer cells and suggest that dual targeting of IGF1R and HER-2 may improve response in HER-2-positive tumours.

Src: a potential target for the treatment of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancers lack expression of estrogen and progesterone receptors and overexpression of human epidermal growth factor receptor 2 (HER2). Unlike other subgroups of patients with breast cancer, targeted therapy is currently unavailable for these patients. The aim of this study was to investigate v-src sarcoma viral oncogene homolog (Src) as a potential target for the treatment of triple-negative breast cancer. METHODS: Expression of Src was measured in 87 triple-negative and 93 non-triple-negative breast cancers. Dasatinib (an inhibitor of Src) was tested in a panel of breast cancer cell lines. RESULTS: Cytoplasmic expression of Src was detected in 83 (95%) triple-negative samples versus 78 (84%) non-triple-negative samples (P = 0.012), while membrane Src was detected in 78% triple-negative compared with 38% of non-triple-negative specimens (P < 0.0001). Dasatinib inhibited growth in three of five triple-negative cell lines (IC(50) < 1 µM). Dasatinib combined with cisplatin was synergistic in the three dasatinib-sensitive cell lines (combination index < 0.9). Dasatinib, in combination with 5'-deoxy-5'-fluoruridine, displayed synergy or additivity. Moderate synergy was observed with docetaxel (Taxotere) in two cell lines but the combination was antagonistic in HCC-1143 cells. CONCLUSIONS: We conclude that dasatinib with cisplatin is a rational drug combination for testing in triple-negative breast cancer.

Tumor markers in pancreatic cancer: a European Group on Tumor Markers (EGTM) status report.

Pancreatic ductal adenocarcinoma is one of the most difficult malignancies to diagnose and treat. The aim of this article is to review how tumor markers can aid the diagnosis and management of patients with this malignancy. The most widely used and best validated marker for pancreatic cancer is CA 19-9. Inadequate sensitivity and specificity limit the use of CA 19-9 in the early diagnosis of pancreatic cancer. In non-jaundiced patients, however, CA 19-9 may complement other diagnostic procedures. In patients with resectable pancreatic cancer, presurgical and postresection CA 19-9 levels correlate with overall survival. In advanced disease, elevated pretreatment levels of CA 19-9 are associated with adverse patient outcome and thus may be combined with other factors for risk stratification. Most, but not all, reports indicate that serial levels of CA 19-9 correlate with response to systemic therapy. Use of CA 19-9 kinetics in conjunction with imaging is therefore recommended in monitoring therapy. Although several potential serum and tissue markers for pancreatic cancer are currently undergoing evaluation, none are sufficiently validated for routine clinical use. CA 19-9 thus remains the serum pancreatic cancer marker against which new markers for this malignancy should be judged.

Estrogen receptor-beta mRNA is associated with adverse outcome in patients with breast cancer.

BACKGROUND/AIMS: Estrogen receptor (ER) is the prototype therapy predictive marker in oncology. The ER is now known to exist in two main forms with similar overall structure: ER-alpha and ER-beta. Both forms may be expressed in breast cancer. The aim of this study was to examine breast cancer outcome in relation to expression of ER-beta. METHODS: In this investigation, we measured the expression of ER-alpha protein and ER-beta mRNA in 121 extracts of invasive breast cancer. Association of expression with clinical outcome was examined using Kaplan-Meier and Cox regression analyses. RESULTS: While ER-alpha expression was associated with good patient outcome [hazard ratio (HR) for death from breast cancer 0.37; 95% confidence interval (CI) 0.17-0.84; p = 0.017], ER-beta predicted poor outcome (HR for death from breast cancer 2.49; 95% CI 1.10-5.63; p = 0.028). CONCLUSION: Based on these findings, we conclude that ER-beta may have a different biological role from that of ER-alpha in breast cancer.

A zinc phthalocyanine-benzoperylenetriimide conjugate for solvent dependent ultrafast energy vs. electron transfer.

A novel zinc phthalocyanine-benzoperylenetriimide conjugate has been synthesized and its ability to undergo ultrafast energy and electron transfer as a function of solvent polarity has been demonstrated using the femtosecond transient absorption (fs-TA) technique operating at a femto- to nanosecond time scale.

Protein kinase Cdelta expression in breast cancer as measured by real-time PCR, western blotting and ELISA.

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCdelta. Currently, it is unclear whether PKCdelta is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCdelta in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cdelta expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCdelta but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCdelta concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCdelta mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cdelta mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann-Whitney U-test). Increasing concentrations of PKCdelta mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCdelta in breast cancer progression.

Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database.

Traditionally, matrix metalloproteinases (MMPs) have been implicated in cancer invasion and metastasis. Because of their role in these processes, several MMPs have been investigated for potential prognostic value as well as targets for antimetastatic therapy. In this investigation, we used a publically available database to relate messenger RNA expression levels for 17 different MMPs to tumor characteristics and outcome in patients with breast cancer. Of the MMPs investigated, only MMP-1 was significantly increased in tumors >2 cm in size compared with those

ADAM-17 predicts adverse outcome in patients with breast cancer.

ADAM-17 is a matrix metalloproteinase-like enzyme involved in the release of several ligands that have been shown to promote both cancer formation and progression. These ligands include transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor, epiregulin and tumor necrosis factor-alpha. In this investigation, we measured the expression of total ADAM-17 by enzyme-linked immunosorbent assay in 153 invasive breast cancers. We also measured the precursor and active forms by western blotting in 140 invasive breast cancers. Expression of ADAM-17 was significantly increased in high-grade compared with low-grade tumors and was independent of tumor size, lymph node metastasis and estrogen receptor status. Patients with high expression of ADAM-17 had a significantly shorter overall survival compared with those with low expression. Significantly, the prognostic impact of ADAM-17 was independent of conventional prognostic factors for breast cancer. Our results are further evidence that ADAM-17 is involved in breast cancer progression and thus provides further impetus for exploiting ADAM-17 as new target for cancer treatment.

Trends in vitamin D supplement use in a general female and breast cancer population in Ireland: A repeated cross-sectional study.

BACKGROUND: Vitamin D has been linked with improved survival after breast cancer diagnosis but little is known about prescribing rates. This study investigates trends in vitamin D supplement use in both a general female and breast cancer population. METHODS: Women with a breast cancer diagnosis were identified from the National Cancer Registry of Ireland (n = 19870). Women who had any vitamin D claim between 2005 and 2011 were identified from pharmacy claims data (n = 8556). Prevalence rates were calculated as a proportion of all eligible women and by age (< 55 years, ≥ 55 years). Poisson regression was used to compare rates of vitamin D prescribing across years (risk ratio (RR), 95% CI). RESULTS: There was a statistically significant increase in women with a claim for vitamin D between 2005-2011, with the largest increase among breast cancer patients aged ≥ 55 years (RR = 2.26; 95% CI, 2.11-2.42). CONCLUSION: This may have significant public health implications if associations between vitamin D and improved breast cancer survival prove to be causal.

Tumour markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines for clinical use.

The aim of this article is to present updated guidelines for the use of serum, tissue and faecal markers in colorectal cancer (CRC). Lack of specificity and sensitivity preclude the use of all existing serum markers for the early detection of CRC. For patients with stage II or stage III CRC who may be candidates for either liver resection or systemic treatment should recurrence develop, CEA should be measured every 2-3 months for at least 3 years after diagnosis. Insufficient evidence exists to recommend routine use of tissue factors such as thymidylate synthase, microsatellite instability (MSI), p53, K-ras and deleted in colon cancer (DCC) for either determining prognosis or predicting response to therapy in patients with CRC. Microsatellite instability, however, may be used as a pre-screen for patients with suspected hereditary non-polyposis colorectal cancer. Faecal occult blood testing but not faecal DNA markers may be used to screen asymptomatic subjects 50 years or older for early CRC.

Clinical use of biomarkers in breast cancer: Updated guidelines from the European Group on Tumor Markers (EGTM).

Biomarkers play an essential role in the management of patients with invasive breast cancer. For selecting patients likely to respond to endocrine therapy, both oestrogen receptors (ERs) and progesterone receptors (PRs) should be measured on all newly diagnosed invasive breast cancers. On the other hand, for selecting likely response to all forms of anti-HER2 therapy (trastuzumab, pertuzumab, lapatinib or ado-trastuzumab emtansine), determination of HER2 expression or gene copy number is mandatory. Where feasible, measurement of ER, PR and HER2 should be performed on recurrent lesions and the primary invasive tumour. Although methodological problems exist in the determination of Ki67, because of its clearly established clinical value, wide availability and low costs relative to the available multianalyte signatures, Ki67 may be used for determining prognosis, especially if values are low or high. In oestrogen receptor (ER)-positive, HER2-negative, lymph node-negative patients, multianalyte tests such as urokinase plasminogen activator (uPA)-PAI-1, Oncotype DX, MammaPrint, EndoPredict, Breast Cancer Index (BCI) and Prosigna (PAM50) may be used to predict outcome and aid adjunct therapy decision-making. Oncotype DX, MammaPrint, EndoPredict and Prosigna may be similarly used in patients with 1-3 metastatic lymph nodes. All laboratories measuring biomarkers for patient management should use analytically and clinically validated assays, participate in external quality assurance programs, have established assay acceptance and rejection criteria, perform regular audits and be accredited by an appropriate organisation.

Ets2 transcription factor in normal and neoplastic human breast tissue.

The Ets family of transcription factors regulate the expression of multiple genes involved in tumour formation and progression. The aim of this work was to test the hypothesis that the expression of Ets2 in breast cancers was associated with parameters of tumour progression and metastasis. Using reverse-transcriptase polymerase chain reaction (RT-PCR), Ets2 mRNA was detected in 69% of 181 breast carcinomas, 63% of 43 fibroadenomas and 47% of 43 specimens of normal breast tissue. Levels were significantly higher in carcinomas compared with normal breast tissue (P = 0.006). Using Western blotting, Ets2 protein was found to migrate as two bands with molecular masses of 52 kDa (p52) and 54kDa (p54). Levels of both proteins were significantly higher in the carcinomas compared with both fibroadenomas (P = 0.0001) and normal breast tissue (P = 0.0001). In the carcinomas, a significant relationship was found between the p52 and p54 form of Ets2 (r = 0.51, P < 0.0001; Spearman correlation). Also, in the carcinomas, a significant correlation was found between both forms of Ets2 protein and urokinase plasminogen activator (uPA) (for p52, r = 0.43, P = 0.0005, n = 68; for p54, r = 0.50, P = 0.0001, n = 68). As Ets2 binding sites are present on the uPA promoter, Ets2 may be one of the transcription factors regulating uPA expression in human breast cancer.

Tissue-type plasminogen activator in breast cancer: relationship with estradiol and progesterone receptors.

Plasminogen activator (PA) is an estradiol-inducible enzyme and therefore a potential marker for a functional estradiol receptor (ER) in human breast carcinomas. In this investigation tissue-type PA (t-PA) correlated significantly with both ER and progesterone receptors (PR) in human breast carcinomas. In contrast, neither total PA activity nor urokinase-like PA showed any significant correlation with either ER or PR. Other proteases such as a trypsin-like protease, a chymotrypsin-like protease, and cathepsin B also showed no correlation with ER and PR. It was concluded that the t-PA form of PA may be a marker for a functional ER in breast carcinoma and thus be of value in predicting hormone-dependent breast cancers.