Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA

Expression of prostate-specific antigen variant MRNA transcripts in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA) is important in tumour detection, monitoring disease progression and tumour recurrence. however, PSA is not a cancerspecific marker as levels can also be elevated in benign prostatic disease. A number of different mRNA transcripts of PSA have also been identified in prostatic tissue, but have not been fully characterized (PSA 424, PSA 525, Schulz transcript). Tissue specimens from transurethral resection of the prostate (TURP) or radical prostatectomy were obtained from 17 men with BPH and 15 men with prostate cancer. Total RNA was extracted, and reverse-transcriptionpolymerase chain reaction (RT-PCR) and Southern analysis carried out using transcript-specific primers and probes to determine which mRNA PSA transcripts were expressed. Real-time PCR was performed to determine transcript levels between the two groups using transcript-specific primers and SYBR green fluorescence. Values obtained were normalized to a standard housekeeping gene, B2-microglobulin. Transcripts amplified by RT-PCR and real-time PCR were confirmed by DNA sequencing. Our results show that the transcripts were present in some, but not all, BPH and cancer samples indicating that they are not specific to either BPH or cancer. Analysis of real-time PCR normalized values using a Student’s t -test, shows that there is a significant difference between the two groups for PSA 424, but not wild-type PSA, PSA 525 or the Schulz transcript. Although a larger cohort of samples is needed to further confirm these results, these findings suggest that mRNA levels of PSA 424 may have some utility as a diagnostic or prognostic marker in prostate cancer detection.

Chronic preoperative pain and psychological robustness predict acute postoperative pain outcomes after surgery for breast cancer

Background - Few epidemiological studies have prospectively investigated preoperative and surgical risk factors for acute postoperative pain after surgery for breast cancer. We investigated demographic, psychological, pain-related and surgical risk factors in women undergoing resectional surgery for breast cancer. Methods - Primary outcomes were pain severity, at rest (PAR) and movement-evoked pain (MEP), in the first postoperative week. Results - In 338 women undergoing surgery, those with chronic preoperative pain were three times more likely to report moderate to severe MEP after breast cancer surgery (OR 3.18, 95% CI 1.45–6.99). Increased psychological ‘robustness’, a composite variable representing positive affect and dispositional optimism, was associated with lower intensity acute postoperative PAR (OR 0.63, 95% CI 0.48–0.82) and MEP (OR 0.71, 95% CI 0.54–0.93). Sentinel lymph node biopsy (SLNB) and intraoperative nerve division were associated with reduced postoperative pain. No relationship was found between preoperative neuropathic pain and acute pain outcomes; altered sensations and numbness postoperatively were more common after axillary sample or clearance compared with SLNB. Conclusion - Chronic preoperative pain, axillary surgery and psychological robustness significantly predicted acute pain outcomes after surgery for breast cancer. Preoperative identification and targeted intervention of subgroups at risk could enhance the recovery trajectory in cancer survivors.

A study of patient-specific prognosis of ovarian cancer

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Men's knowledge and attitudes about cervical cancer screening in Kenya.

BACKGROUND: A number of studies have identified male involvement as an important factor affecting reproductive health outcomes, particularly in the areas of family planning, antenatal care, and HIV care. As access to cervical cancer screening programs improves in resource-poor settings, particularly through the integration of HIV and cervical cancer services, it is important to understand the role of male partner support in women's utilization of screening and treatment. METHODS: We administered an oral survey to 110 men in Western Kenya about their knowledge and attitudes regarding cervical cancer and cervical cancer screening. Men who had female partners eligible for cervical cancer screening were recruited from government health facilities where screening was offered free of charge. RESULTS: Specific knowledge about cervical cancer risk factors, prevention, and treatment was low. Only half of the men perceived their partners to be at risk for cervical cancer, and many reported that a positive screen would be emotionally upsetting. Nevertheless, all participants said they would encourage their partners to get screened. CONCLUSIONS: Future interventions should tailor cervical cancer educational opportunities towards men. Further research is needed among both men and couples to better understand barriers to male support for screening and treatment and to determine how to best involve men in cervical cancer prevention efforts.

Breast cancer outcomes following predictive BRCA testing in Northern Ireland

Objectives: Since 1995, BRCA testing has identified 445 women in Northern Ireland who carry a pathogenic BRCA1/2 mutation, without breast cancer (bca) at testing. This study examined outcomes with reference to management, bca risk, and incidence following positive predictive testing. Methods: Patients were identified from the regional genetics database. Electronic clinical records were used to obtain management and outcome details. Median follow-up was to bca diagnosis, risk-reducing mastectomy (rrm), death, or last follow-up. Results: 169 women had a BRCA1 mutation, and 276 BRCA2. ■ BRCA1 cohort: Median follow-up post-testing was 3 years. 56 Women (33%) had rrm, and 12 are awaiting rrm (total 68, 40%) at a median age of 36 years. 12 Women (7%) developed bca, at a median of 2 years following testing. 4 Women were diagnosed with bcas incidentally at rrm. 7 Patients had bilateral mastectomies following a cancer diagnosis. 1 Woman developed bca following rrm (1.7%). Three deaths were reported: 1 breast cancer (1.7%), 1 ovarian cancer (1.7%), and 1 with no recorded breast/ovarian cancer diagnosis. ■ BRCA2 cohort: Median follow-up post-testing was 6 years. rrm was carried out in 75 women (27%), with 20 awaiting rrm (total 95, 35%); median age: 39 years. 16 Women developed bca (5.8%), at a median of 5 years from testing. 6 Women were diagnosed with cancer incidentally at rrm; 9 women had bilateral mastectomy following diagnosis, and 1 developed bca following rrm (1.3%). Five deaths were reported: 1 bca, 1 ovarian cancer, and 3 with no recorded breast/ovarian cancer diagnosis. Conclusions: The uptake of rrm following predictive BRCA testing in Northern Ireland is comparable with that reported elsewhere. The incidence of bca following rrm is low (<2%) in our cohort, with low breast and ovarian cancer–specific mortality following positive predictive testing.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

Preclinical evaluation of a MYCN-specific inhibitor for the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, characterized by rapid growth, early metastasis and acquired drug resistance. SCLC is usually sensitive to initial treatment, however, most patients relapse within few months; thus more effective therapies are urgently needed. Key genetic alterations very frequently observed in SCLC include loss of TP53 and RB1 and mutations in the MYC family genes (MYC, MYCL or MYCN). One of them is amplified and overexpressed in a mutually exclusive manner and represents the most prominent activating oncogene alteration in this malignancy. In particular, MYCN amplification is associated with tumor progression, treatment failure and poor prognosis. Given the role of MYCN in SCLC and its restricted expression profile, MYCN represents a promising therapeutic target; although it is considered undruggable by traditional approaches. An innovative approach to target the oncogene concerns specific MYCN expression inhibition, acting directly at the level of DNA, through an antigene peptide nucleic acid (agPNA) oligonucleotide, called BGA002. This thesis focused on the study of BGA002, as a possible targeted therapeutic strategy for the treatment of MYCN-related SCLC. In this context, BGA002 proved to be a specific and highly effective inhibitor. Furthermore, MYCN silencing induced alterations in many downstream pathways and led to apoptosis, in concomitance with autophagy reactivation. Moreover, systemic administration of BGA002 was effective in vivo as well, significantly increasing survival in MNA mouse models, even in the scenario of multidrug-resistance. In addition, BGA002 treatment successfully reduced N-Myc protein expression and, more importantly, caused a massive diminishment in tumor vascularization in the multidrug-resistant model. Overall, these results proved that MYCN inhibition by BGA002 may represent a new promising precision medicine approach, to treat MYCN-related SCLC.

Molecular engineering of the m13 phage for targeted photodynamic cancer treatment

This thesis explores the advancement of cancer treatment through targeted photodynamic therapy (PDT) using bioengineered phages. It aims to harness the specificity of phages for targeting cancer-related receptors such as EGFR and HER2, which are pivotal in numerous malignancies and associated with poor outcomes. The study commenced with the M13EGFR phage, modified to target EGFR through pIII-displayed EGFR-binding peptides, demonstrating enhanced killing efficiency when conjugated with the Rose Bengal photosensitizer. This phase underscored phages' potential in targeted PDT. A breakthrough was achieved with the development of the M137D12 phage, engineered to display the 7D12 nanobody for precise EGFR targeting, marking a shift from peptide-based to nanobody-based targeting and yielding better specificity and therapeutic results. The translational potential was highlighted through in vitro and in vivo assays employing therapeutic lasers, showing effective, specific cancer cell killing through a necrotic mechanism. Additionally, the research delved into the interaction between the M13CC phage and colon cancer models, demonstrating its ability to penetrate and disrupt cancer spheroids only upon irradiation, indicating a significant advancement in targeting cells within challenging tumor microenvironments. In summary, the thesis provides a thorough examination of the phage platform's efficacy and versatility for targeted PDT. The promising outcomes, especially with the M137D12 phage, and initial findings on a HER2-targeting phage (M13HER2), forecast a promising future for phage-mediated, targeted anticancer strategies employing photosensitizers in PDT.