Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered. The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented. These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.

Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with 211At and 131I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using 124I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies. These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

published_or_final_version / Surgery / Master / Master of Philosophy

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used as an agent for treating ESCC patients undergoing chemotherapy. However establishment of resistance over the course of treatment diminishes the clinical usefulness and is one reason explaining poor prognosis of ESCC patients. In order to gain insights into the mechanism of cisplatin resistance in ESCC, HPLC/nESI-MS/MS proteomic profiling was employed to examine the global protein alterations of cisplatin-resistant ESCC cell line HKESC2/CDDP comparing with its parental cisplatin-sensitive cell line HKESC2. Stable over-expression and knocked-down cell lines were established for pathway analysis and functional studies. Seventeen proteins were identified with more than 2-fold difference in expression levels. These proteins are involved in endoplasmic reticulum stress response, metabolic processes, DNA replication and repair, nucleotide binding and cell cycle control, while some of them are components of cytoskeletal proteins. Among them, 14-3-3σ was one of the most significantly upregulated proteins found in HKESC2/CDDP cells and its differential expression levels were validated using western blotting and real-time quantitative polymerase chain reaction. Pathway analysis revealed that ectopic overexpression of 14-3-3σ caused a general upregulation in DNA repairing genes. Furthermore, functional validation showed that elevated 14-3-3σ expression contributed considerably to the observed cisplatin resistance in HKESC2/CDDP cells. While knocking down 14-3-3σ expression reversed the above situations in SLMT1 cells. I conclude that up-regulation in 14-3-3σ, together with DNA repairing genes, contributes to the establishment of cisplatin resistance in HKESC2/CDDP cells. Knocking down 14-3-3σ expression sensitized ESCC cells to cisplatin treatment, and hence, opens a therapeutic opportunity for ESCC cisplatin resistance. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2010.

p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.

p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.