Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer.

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

KSR1 regulates BRCA1 degradation and inhibits breast cancer growth.

Kinase suppressor of Ras-1 (KSR1) facilitates signal transduction in Ras-dependent cancers, including pancreatic and lung carcinomas but its role in breast cancer has not been well studied. Here, we demonstrate for the first time it functions as a tumor suppressor in breast cancer in contrast to data in other tumors. Breast cancer patients (n>1000) with high KSR1 showed better disease-free and overall survival, results also supported by Oncomine analyses, microarray data (n=2878) and genomic data from paired tumor and cell-free DNA samples revealing loss of heterozygosity. KSR1 expression is associated with high breast cancer 1, early onset (BRCA1), high BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels. Phospho-profiling of major components of the canonical Ras-RAF-mitogen-activated protein kinases pathway showed no significant changes after KSR1 overexpression or silencing. Moreover, KSR1 stably transfected cells formed fewer and smaller size colonies compared to the parental ones, while in vivo mouse model also demonstrated that the growth of xenograft tumors overexpressing KSR1 was inhibited. The tumor suppressive action of KSR1 is BRCA1 dependent shown by 3D-matrigel and soft agar assays. KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance. These data link these proteins in a continuum with clinical relevance and position KSR1 in the major oncoprotein pathways in breast tumorigenesis.

Assessment of safety and interference issues of radio frequency identification devices in 0.3 Tesla magnetic resonance imaging and computed tomography.

The objective of this study was to evaluate two issues regarding magnetic resonance imaging (MRI) including device functionality and image artifacts for the presence of radio frequency identification devices (RFID) in association with 0.3 Tesla at 12.7 MHz MRI and computed tomography (CT) scanning. Fifteen samples of RFID tags with two different sizes (wristband and ID card types) were tested. The tags were exposed to several MR-imaging conditions during MRI examination and X-rays of CT scan. Throughout the test, the tags were oriented in three different directions (axial, coronal, and sagittal) relative to MRI system in order to cover all possible situations with respect to the patient undergoing MRI and CT scanning, wearing a RFID tag on wrist. We observed that the tags did not sustain physical damage with their functionality remaining unaffected even after MRI and CT scanning, and there was no alternation in previously stored data as well. In addition, no evidence of either signal loss or artifact was seen in the acquired MR and CT images. Therefore, we can conclude that the use of this passive RFID tag is safe for a patient undergoing MRI at 0.3 T/12.7 MHz and CT Scanning.

Alcohol use and HIV sexual risk among MSM in Chennai, India.

Men who have sex with men (MSM) in India are a core risk group for HIV. Heavy alcohol consumption is associated with increased sexual risk-taking behaviours in many cultures, in particular among MSM. However, no studies to date have explored alcohol use and HIV risk among MSM in India. MSM in Chennai, India (n = 210) completed an interviewer-administered behavioural and psychosocial assessment. Bivariate and multivariable logistic regression procedures examined behavioural and demographic associations with weekly alcohol consumption. Twenty-eight percent of the sample (n = 58) reported using alcohol at least weekly to the point of being buzzed/intoxicated, which was associated with older age, being married to a woman, being panthi (masculine appearing, predominantly insertive partners) versus kothi (feminine acting/appearing and predominantly receptive partners), weekly tobacco use, unprotected anal sex and unprotected vaginal sex in the three months prior to study enrollment (all P < 0.05). In a multivariable model, unprotected vaginal sex in the previous three months and being married to a women were unique variables associated with weekly alcohol use (all P < 0.01). Further investigation of alcohol use within the context of sexual risk taking is warranted among Indian MSM. Panthis and MSM who are married to women may be particularly likely to benefit from interventions to decrease alcohol intake and concurrent unsafe sex.

The DEAD-box protein p72 regulates ERalpha-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERalpha-positive breast cancer.

The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17) have been shown to act as transcriptional co-activators for a diverse range of transcription factors, including oestrogen receptor-alpha (ERalpha). Here, we show that, although both proteins interact with and co-activate ERalpha in reporter gene assays, small interfering RNA-mediated knockdown of p72, but not p68, results in a significant inhibition of oestrogen-dependent transcription of endogenous ERalpha-responsive genes and oestrogen-dependent growth of MCF-7 and ZR75-1 breast cancer cells. Furthermore, immunohistochemical staining of ERalpha-positive primary breast cancers for p68 and p72 indicate that p72 expression is associated with an increased period of relapse-free and overall survival (P=0.006 and 0.016, respectively), as well as being inversely associated with Her2 expression (P=0.008). Conversely, p68 shows no association with relapse-free period, or overall survival, but it is associated with an increased expression of Her2 (P=0.001), AIB-1 (P<0.001) and higher tumour grade (P=0.044). Our data thus highlight a crucial role for p72 in ERalpha co-activation and oestrogen-dependent cell growth and provide evidence in support of distinct but important roles for both p68 and p72 in regulating ERalpha activity in breast cancer.