A Review: Genetic Mutations as a Key to Unlocking Drug Resistance in Cervical Cancer.

Cervical cancer is the fourth most common cancer in women. Advanced stage and metastatic disease are often associated with poor clinical outcomes. This substantiates the absolute necessity for high-throughput diagnostic and treatment platforms that are patient and tumour specific. Cervical cancer treatment constitutes multimodal intervention. Systemic treatments such as chemotherapy and/or focal radiotherapy are typically applied as neoadjuvant and/or adjuvant strategies. Cisplatin constitutes an integral part of standard cervical cancer treatment approaches. However, despite initial patient response, de novo or delayed/acquired treatment resistance is often reported, and toxicity is of concern. Chemotherapy resistance is associated with major alterations in genomic, metabolomic, epigenetic and proteomic landscapes. This results in imbalanced homeostasis associated with pro-oncogenic and proliferative survival, anti-apoptotic benefits, and enhanced DNA damage repair processes. Although significant developments in cancer diagnoses and treatment have been made over the last two decades, drug resistance remains a major obstacle to overcome.

Despite advances in treatment, the disease's advanced stages and spread to other parts of the body often lead to poor outcomes. This highlights the urgent need for better diagnostic and treatment methods tailored to each patient and their specific tumour. Treatment for cervical cancer usually involves a combination of therapies. Chemotherapy and focused radiation therapy are commonly used before or after surgery to improve outcomes. However, some patients develop resistance to these treatments, either from the start or after initially responding to therapy. This resistance can make treatment less effective and increase the risk of side effects. Chemotherapy resistance is often linked to changes in the genes and proteins of cancer cells. These changes disrupt the normal balance within the cells, making them more prone to grow and survive, resist cell death, and repair DNA damage caused by treatment. Despite progress in cancer research and treatment, drug resistance remains a significant challenge. This review aims to explore how acquired genetic mutations contribute to drug resistance in cervical cancer. By understanding these mutations better, researchers and clinicians in low- to middle-income countries can develop more effective treatment strategies to improve outcomes for patients.

Highlighting the role of CD44 in cervical cancer progression: immunotherapy's potential in inhibiting metastasis and chemoresistance.

Cervical cancer affects thousands of women globally with recurring high-risk HPV infections being at the centre of cervical pathology. Oncological treatment strategies are continually challenged by both chemoresistance and metastasis within patients. Although both work hand-in-hand, targeting their individual mechanisms could prove highly beneficial for treatment outcomes. Such targets include the metastatic-promoting stem cell marker, CD44, which is abundant in cervical cancer cells and is common to both chemoresistance and metastatic mechanisms. Seeing that many existing advanced-stage cervical cancer treatment regimes, such as platinum-based chemotherapy regimens, remain limited and are rarely curative, alternative treatment options within the field of immunology are being considered. The use of immune checkpoint inhibition therapy, which targets immune checkpoints, CTLA-4 and PD-1/PD-L1, has shown promise as an alternate standard of care for patients suffering from advanced-stage cervical cancer. Therefore, this review aims to assess whether immune checkpoint inhibition can mitigate the pathological effects of CD44-induced EMT, metastasis, and chemoresistance in cervical cancer patients.

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

BACKGROUND: The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). METHODS: Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. RESULTS: Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. CONCLUSIONS: The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks.

BACKGROUND AND PURPOSE: IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. METHODS: We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. RESULTS: We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30-40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. CONCLUSIONS: These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C.

BACKGROUND: Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. RESULTS: During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP).To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor.We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under ß-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. CONCLUSIONS: This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important regulator of cardiac contractility. This has further implications for understanding the patho-aetiology of HCM-causing mutations in the genes encoding cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing or modifying factor.

The EGF receptor interacts with the type 1 IGF receptor and regulates its stability.

Both the epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF1R) require homo- and hetero-dimerisation with their own family members to acquire full function. We recently showed that IGF1R gene silencing led to EGFR hyper-phosphorylation in human breast cancer cells, and hypothesised that this crosstalk might be associated with direct IGF1R:EGFR interaction. Indeed we could detect reciprocal co-precipitation between the IGF1R and EGFR when overexpressed in SKUT-1 cells, and between endogenous IGF1R and EGFR in MDA-MB-468 breast carcinoma cells, two squamous cancer cell lines, and clinical samples of breast cancer. Interaction was abolished by knockdown of either receptor, and we noted that EGFR knockdown also suppressed IGF1R protein levels. Further investigation revealed that EGFR depletion induced enhancement of IGF1R ubiquitylation and degradation. These results indicate novel evidence of crosstalk between two key cancer treatment targets, capable of modifying the stability of IGF1R protein.

Dual silencing of the EGF and type 1 IGF receptors suggests dominance of IGF signaling in human breast cancer cells.

Signaling via the type 1 insulin-like growth factor receptor (IGF1R) confers resistance to EGF receptor (EGFR) inhibitors. It is plausible that reciprocal EGFR compensation could mediate resistance to IGF1R inhibition, prompting us to investigate effects of IGF1R depletion on EGFR signaling in breast cancer cells expressing relatively high (MDA-MB-468) or low (MCF7) EGFR. Transient IGF1R knockdown induced enhanced phosphorylation of the EGFR and its effectors JNK, ERKs and STAT5, but this did not prevent apoptosis induction and inhibition of clonogenic survival following IGF1R knockdown. We used IGF1R shRNA to induce chronic IGF1R depletion, and achieved stable gene silencing in MCF-7 cells; here, EGFR overexpression led to EGFR hyperphosphorylation, again without abrogating survival inhibition after IGF1R knockdown. In both cell lines, dual receptor knockdown prevented EGFR hyperphosphorylation, but induced no greater inhibition of clonogenic survival than IGF1R knockdown alone. These results suggest that the EGFR cannot compensate for IGF1R depletion, and are encouraging for the strategy of IGF1R targeting.

Silencing of the IGF1R gene enhances sensitivity to DNA-damaging agents in both PTEN wild-type and mutant human prostate cancer.

The type 1 insulin-like growth factor receptor (IGF1R) is overexpressed in prostate cancer, and mediates proliferation, motility, and survival. Many prostate cancers harbor inactivating PTEN mutations, enhancing Akt phosphorylation. This activates the principal antiapoptotic pathway downstream of the IGF1R, calling into question the value of IGF1R targeting in this tumor. The aim of the current study was to assess the effect of IGF1R gene silencing in prostate cancer cells that lack functional PTEN protein. In human DU145, LNCaP and PC3 prostate cancer cells, transfection with IGF1R small interfering RNA induced significant enhancement of apoptosis and inhibition of survival, not only in PTEN wild-type DU145 but also in PTEN mutant LNCaP and PC3. This was attributed to attenuation of IGF signaling via Akt, ERKs and p38. In both DU145 and PC3, IGF1R knockdown led to enhancement of sensitivity to mitoxantrone, etoposide, nitrogen mustard and ionizing radiation. There was no sensitization to paclitaxel or 5-fluorouracil, which do not damage DNA, suggesting that chemosensitization results from impairment of the DNA damage response, in addition to removal of apoptosis protection. These results support the concept of IGF1R targeting in prostate cancer, and indicate that PTEN loss does not render tumor cells refractory to this strategy.

A simple and cost-effective method for producing small interfering RNAs with high efficacy.

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs.

The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.

The type 1 insulin-like growth factor receptor (IGF1R) is often overexpressed by tumors and mediates growth and apoptosis protection. We previously showed that antisense reagents complementary to the IGF1R translation start site enhance radio- and chemosensitivity and impair Atm function. However these agents induce relatively modest IGF1R down-regulation and affect insulin receptor levels. To identify alternative sites for molecular targeting, we utilized scanning oligonucleotide arrays to probe the secondary structure of IGF1R mRNA. This strategy enabled selection of antisense oligonucleotides that generated high heteroduplex yield with IGF1R but not insulin receptor transcripts. Antisense oligonucleotides that hybridized strongly to IGF1R mRNA caused IGF1R down-regulation within intact tumor cells, whereas weakly hybridizing oligonucleotides were inactive. Furthermore, the ability of small interfering RNAs (siRNAs) to block IGF1R expression correlated with the accessibility of the target sequence within the transcript. Thus, siRNAs corresponding to weakly hybridizing oligonucleotides caused minor IGF1R down-regulation, whereas siRNAs homologous to accessible targets induced profound sequence-specific IGF1R gene silencing, blocked IGF signaling, and enhanced tumor cell radiosensitivity. This indicates that secondary structure in the target transcript has a major effect on siRNA efficacy. These findings have implications for siRNA design and suggest that IGF1R-targeting agents incorporating this mode of action have potential as anticancer therapy.