TRF2 Overexpression at the Surgical Resection Margin: A Potential Predictive Biomarker in Oral Squamous Cell Carcinoma for Recurrence.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers in India with high incidence rate in eastern region due to habits of tobacco, pan and gutkha chewing habits. In majority of OSCC, the cases were presented to clinicians at later stages of the disease which leads to increased mortality. In addition presence of minimal residual disease also significantly contributed towards disease progression. Therefore, identification of potential biomarker for prognostic stratification of patients with high risk of disease recurrence and appropriate management is utmost necessary. In this study, 80 OSCC patients were included and their tumour specimen along with cut margin (CM) was collected after surgical excision. Immunohistochemistry (IHC) was performed to check expression of TRF2 in tumour and CM of OSCC patients. Statistical analysis was carried out using SPSS based on clinical and pathological records. It was observed that 27 OSCC patients developed recurrence during the period of the study (2012-2016). It was observed that, in 34 cases (42.25%) TRF2 expression was positive in tumour, while in 46 cases (57.75%), it was negative, while it was just reverse at CM, respectively. The odds of recurrence among patients having high levels of TRF2 in CM were 2.6 times higher than the odds of recurrence among patients having lower levels of TRF2 in CM. In conclusion, this study showed that TRF2 at surgical cut margin has a prognostic significance and can be used as a molecular marker for predicting survival in OSCC patients.

𝛽-Catenin-a Possible Prognostic Molecular Marker for Recurrence in Histopathologically Negative Surgical Margin of Oral Cancer.

The locoregional recurrence in oral cancer is not predicted by the histopathological parameters solely as the normal morphological looking cells harbor the genomic instability which acts as the potential tumor cells for recurrence in future. Therefore, there is an urgent need of the biomarker for prognostic stratification of patients with high risk of disease recurrence and appropriate management. Eighty oral squamous cell carcinoma (OSCC) patients were included in the study during the period 2012 to 2014 at Apollo Hospitals and Kalinga Institute of Medical sciences, Bhubaneswar. OSCC tissue samples were collected at the time of surgical excision, and immunohistochemistry (IHC) was performed to check the expression of ß-catenin in cut margin (CM) and tumor. Statistical analysis was carried out using SPSS based on clinical and pathological records. It was observed that among 80 patients, 33.75% (27 patients) developed recurrence. The recurrence rate was low for 6 out of 27 patients (22.2%) where ß-catenin is positive in tumor and negative in cut margin, while it was quite high in 21 out of 27 (77.8%) when marker is negative in tumor but positive in cut margin (CM). The odds of recurrence among patients having high levels of 𝛽-catenin in CM was 3.6 times higher than the odds of recurrence among patients having lower levels of 𝛽-catenin in CM (p < 0.017). In conclusion, this study highlighted that 𝛽-catenin can be included as a prognostic molecular marker, along with routine histopathological study to influence therapeutic decisions and appropriate management of disease.

p38 Mitogen-activated protein kinase modulates cisplatin resistance in Head and Neck Squamous Cell Carcinoma cells.

OBJECTIVE: This study aims to investigate the role of p38 Mitogen-activated protein kinase (MAPK) in imparting cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) cells. DESIGN: Laboratory generated cisplatin resistant HNSCC cells were treated with p38 inhibitor and were subjected to increasing dosage of cisplatin. Western blot, immunohistochemistry and RT PCR analysis were performed to investigate expression level of p-p38 and Cancer stem cell (CSC) markers in cisplatin resistant HNSCC cells with or without p38 inhibitor. Chemoresistance, wound healing capacity and Spheroids formation capacity were assessed following p38 inhibition in cisplatin resistant HNSCC cell lines. In addition, alkaline comet assay and γ-H2AX immunostaining were performed to evaluate the DNA damage response and repair abilities in cisplatin resistant HNSCC cells after p38 inhibition. RESULTS: It was observed that following p38 inhibition, cisplatin resistant HNSCC cells exhibited significant reduction in expression of CSC markers, ß-catenin, reduced migration potential and sphere forming ability along with increased apoptotic index demonstrating there was increased sensitivity towards Cisplatin. Molecular docking study identified several interface amino acid residues between p-p38 with CSC markers (Klf4 and CD44). p38 inhibited cisplatin resistant HNSCC cells also exhibited increased DNA damage as measured by Comet assay and γ-H2AX foci formation index. There was significant decrease in DNA repair as confirmed by reduced ERRC1 expression. CONCLUSIONS: Our study demonstrated that p38 MAPK inhibition can be a targeted approach to overcome resistance in HNSCC thereby escalating the effectiveness of chemotherapy in HNSCC.

Role of telomeric RAP1 in radiation sensitivity modulation and its interaction with CSC marker KLF4 in colorectal cancer.

Aims: Radiotherapy is predominantly used as one of the treatment modalities to treat local tumor in colorectal cancer (CRC). Hindrance in disease treatment can be attributed to radio-tolerance of cancer stem cells (CSCs) subsistence in the tumor. Understanding the radio-resistant property of CSCs might help in the accomplishment of targeted radiotherapy treatment and increased disease-free survival. Telomeric RAP1 contributes in modulation of various transcription factors leading to aberrant cell proliferation and tumor cell migration. Therefore, we investigated the role of RAP1 in maintaining resistance phenotype and acquired stemness in radio-resistant cells.Main methods: Characterization of HCT116 derived radio-resistant cell (HCT116RR) was performed by cell survival and DNA damage profiling. RAP1 silenced cells were investigated for DNA damage and expression of CSC markers through western blotting and Real-time PCR post-irradiation. Molecular docking and co-immunoprecipitation study were performed to investigate RAP1 and KLF4 interaction followed by RAP1 protein status profiling in CRC patient.Key findings: We established radio-resistant cells, which showed tolerance to radiotherapy and elevated expression of CSC markers along with RAP1. RAP1 silencing showed enhanced DNA damage and reduced expression of CSC markers post-irradiation. We observed strong physical interaction between RAP1 and KLF4 protein. Furthermore, higher RAP1 expression was observed in the tumor of CRC patients. Dataset analysis also revealed that high expression of RAP1 expression is associated with poor prognosis.Significance: We conclude that higher expression of RAP1 implicates its possible role in promoting radio-resistance in CRC cells by modulating DNA damage and CSC phenotype.

Inhibition of CD44 sensitizes cisplatin-resistance and affects Wnt/ß-catenin signaling in HNSCC cells.

CD44 is one of the key cancer stem-like cell (CSC) marker and may have a potential role in tumorigenesis. In this study, we investigated the role of CD44 in prognosis of HNSCC patients, its possible crosstalk with Wnt/ß-catenin signaling and modulating cisplatin resistance. We observed increased expression of CD44 in the cut margin of recurrent HNSCC patients were associated with poor prognosis. We observed that inhibition of CD44 by using 1,2,3,4 tetrahydroisoquinoline (THIQ) modulates the expression of Wnt/ ß-catenin signaling proteins and further silencing of ß-catenin also decreases the expression of CD44. This led us to investigate the possible protein-protein interaction between CD44 and ß-catenin. Co-immunoprecipitation study illustrated possible interaction between CD44 and ß-catenin which was further confirmed by molecular docking and molecular dynamic (MD) simulation studies. Molecular docking study revealed that one interface amino acid residue Glu642 of ß -catenin interacts with Lys92 of CD44 which was also present for 20% of simulation time. Furthermore, we observed that inhibition of CD44 chemosensitizes cisplatin-resistant HNSCC cells towards cisplatin. In conclusion, this study investigated the possible role of CD44 along with Wnt/ ß-catenin signaling and their possible therapeutic role to abrogate cisplatin resistance.

Nonhistone human chromatin protein PC4 is critical for genomic integrity and negatively regulates autophagy.

Multifunctional human transcriptional positive co-activator 4 (PC4) is a bona fide nonhistone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional genome organization. Knockdown of PC4 expression causes a drastic decompaction which leads to open conformation of the chromatin, and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation, possibly through enhanced autophagic activity. Moreover, PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the ability of PC4 knockdown cells to survive. On the contrary, complementation with wild-type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in the absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during stress conditions such as radiation.

A Child with Partial Trisomy 4 (q26 - qterminal) Resulting from Paternally Inherited Translocation (4:18) Associated with Multiple Congenital Anomalies and Death.

Parental balanced reciprocal translocations can result in partial aneuploidy in the offspring due to unbalanced meiotic segregation during gametogenesis. Herein, we report the phenotypic and cytogenetic characterization in a 9-day-old male child with partial trisomy of chromosome 4. Karyotyping of the proband and parents was performed along with multicolor fluorescence in situ hybridization (mFISH) of paternal chromosomes. Conventional cytogenetic analysis by karyotyping showed 47,XY,der(18),t(4;18)(q26;q22),+4 in proband, and the paternal karyotype was found as 47,XY,der(18),t(4;18)(q26;q22). mFISH analysis on paternal chromosomal preparations confirmed both region and origin of the balanced translocation. In this study, karyotyping helped us to identify both numerical and structural anomalies in the proband, and mFISH helped us to confirm our cytogenetic findings. Therefore, cytogenetic screening of both partners is recommended before pregnancy to rule out or confirm the presence of any numerical or structural anomaly in one, both, or none of the partners.

KLF4 expression in the surgical cut margin is associated with disease relapse of oral squamous cell carcinoma.

OBJECTIVE: The presence of cancer stem-like cells (CSCs) in the majority of tumors is one of the factors responsible for disease relapse in oral squamous cell carcinoma (OSCC). In this study, we investigated the role of octamer-binding transcription factor 4 (OCT4) and Kruppel-like factor 4 (KLF4) in OSCC progression and disease relapse. STUDY DESIGN: In this study, 102 patients with OSCC were included. The expression of ß-catenin and CSC markers (KLF4 and OCT4) in surgical cut margin and tumor were investigated through Western blot analysis, immunohistochemistry, and quantitative polymerase chain reaction analysis. The χ2 test was used to evaluate the association of ß-catenin, OCT4, and KLF4 expression with clinicopathologic characteristics. Kaplan-Meier and Cox regression analyses were performed to correlate different clinical factors with the prognoses of patients with OSCC. RESULTS: We observed increased expression of OCT4, KLF4, and ß-catenin in the cut margins (CMs) in recurrent OSCC. The χ2 test exhibited recurrence as one of the key factors associated with high expression of these markers. Kaplan-Meier and COX regression analyses demonstrated that increased expression of KLF4 in the CM region of recurrent patients was independently associated with a poor prognosis. CONCLUSIONS: Our findings indicated that expression of KLF4 can be used for monitoring disease progression and may serve as prognostic marker to predict recurrence.

p38 MAPK pathway and its interaction with TRF2 in cisplatin induced chemotherapeutic response in head and neck cancer.

TRF2 is a telomere binding protein, a component of the shelterin complex that plays a major role in maintaining the integrity of the genome. TRF2 is over-expressed in a number of human cancers including Head and Neck cancer and might play a key role in tumor initiation and development. p38 MAPK signaling pathway is strongly activated in response to various environmental and cellular stresses and thus overexpressed in most of the Head and Neck cancer cases. In this study, we investigated potential interactions of TRF2 with p38 in HNSCC cells and patient samples. Using in silico experiments, we identified interface polar residue Asp-354 of p38 and Arg-492, Arg-496 of TRF2 as protein-protein interaction hotspots. In addition to these interactions, Arg-49 residue of p38 was also found to interact with Glu-456 of TRF2. A detailed understanding of how phosphorylated and unphosphorylated state of p38 protein can influence the stability, specificity and to some extent a conformational change of p38-TRF2 binding is presented. Silencing of TRF2 significantly decreased the phosphorylation of p38 in HNSCC cells which was confirmed by western blot, immunofluorescence and co-immunoprecipitation and alternatively inhibiting p38 using p38 inhibitor (SB 203580) decreased the expression of TRF2 in HNSCC cells. Furthermore, we checked the effect of TRF2 silencing and p38 inhibition in cisplatin induced chemosensitivity of SCC-131 cells. TRF2 silencing and p38 inhibition chemosensitize HNSCC cells to cisplatin. Thus, targeting TRF2 in combinatorial therapeutics can be a treatment modality for Head and Neck cancer which involves inhibition of p38 MAPK pathway.

Role of Telomeric TRF2 in Orosphere Formation and CSC Phenotype Maintenance Through Efficient DNA Repair Pathway and its Correlation with Recurrence in OSCC.

The major problem to effective treatment of oral cancer is the presence of therapy resistance. Presence of cancer stem cell in the bulk of tumor have been implicated in therapeutic resistance. In this study, we report a non-telomeric role of TRF2 in formation of oral cancer spheroids and CSC phenotype maintenance via an efficient DNA damage repair mechanism in the presence of chemotherapeutic insult. We report reduced sphere formation efficiency and reduced spheroid size in TRF2 silenced oral cancer cell lines. TRF2 silenced orospheres further reported reduced proliferative capacity as compared to non-silenced orospheres. Furthermore, TRF2 silencing hampered the migratory potential of oral cancer cell line and also reduced the expression of several CSC markers like CD44, Oct4, Sox2, KLF4 and c-Myc along with ß-catenin and hTERT molecules both in Cal27 cell line and generated orospheres. TRF2 silencing impaired efficient DNA damage repair capacity of non-orospheric and orospheric cells and repressed ERCC1 expression levels when treated with Cisplatin. TRF2 overexpression was also observed to correlate with poor overall survival and disease relapse of OSCC patients. In silico studies further identified several amino acid residues that show high binding affinity and strong protein-protein interactions among TRF2 and CSC marker KLF4. Hence, our report confirms a non-telomeric role of TRF2 in spheroid generation, maintenance of CSC phenotype and efficient DNA damage repair capacity contributing to chemotherapy resistance in oral cancer cell line. We further iterate the use of TRF2 as a prognostic marker in OSCC for faster detection and improved survival.

Role of p38 MAPK in disease relapse and therapeutic resistance by maintenance of cancer stem cells in head and neck squamous cell carcinoma.

AIM: This study aimed to investigate the role of p38 MAPK in maintenance of cancer stem cell (CSC) phenotype, therapy resistance, and DNA damage repair and response in head and neck squamous cell carcinoma (HNSCC). METHODS: In this study, 104 HNSCC patients were included. Western blot, immunohistochemistry, and qPCR analysis were performed to investigate the expression level of p-p38 and CSC markers in cut margin and tumor area of HNSCC patients. The expression level of p-p38 and CSC markers was also evaluated in HNSCC cells with or without p38 inhibitor. Chemoresistance, wound healing capacity, and multicellular tumor spheroids (MCTS) formation capacity were evaluated in HNSCC-derived cell lines with or without p38 inhibitor. In addition, DNA damage response and repair capacities were also evaluated in HNSCC cells after p38 inhibition using alkaline comet assay and γ-H2 AX immunostaining. RESULT: We observed that recurrence could be associated with upregulated status of p-p38 and p38α gene in cut margin area of HNSCC patients as compared to tumor region. p38-inhibited cells showed significantly reduced expression of CSC markers, chemosensitivity toward cisplatin, reduced migration potential, and sphere-forming ability along with increased apoptotic population after treatment with increasing concentration of cisplatin. p38-inhibited cells also exhibited significantly increased comet olive tail moment and accumulation of γ-H2 AX, demonstrating increased DNA damage. CONCLUSION: This study demonstrated that p38 MAPK activation may play a role in therapeutic resistance and disease relapse in HNSCC by maintenance of CSCs phenotype.

Role of ß-catenin in cisplatin resistance, relapse and prognosis of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer in India with high incidence and rapid recurrence rates. Here, we aimed to investigate the role of ß-catenin, a developmental pathway gene, in HNSCC therapy resistance, DNA damage response, recurrence and prognosis. METHODS: In total 80 HNSCC samples were included. Western blot, immunohistochemistry and qRT-PCR analyses were performed to assess ß-catenin expression in the cut margin and tumor areas of each sample. Kaplan-Meier analyses were performed to correlate ß-catenin expression with the survival and prognosis of HNSCC patients. In addition, chemo-resistance, DNA damage response and DNA repair capacities were evaluated in HNSCC-derived cell lines through LiCl-mediated up-regulation and siRNA-mediated silencing of ß-catenin expression. RESULTS: We observed ß-catenin up-regulation in cut margin areas of recurrent patients compared to their corresponding tumor regions, which subsequently could be associated with poor prognosis. In addition, we found that LiCl-mediated up-regulation of ß-catenin in HNSCC-derived cells led to cisplatin resistance, evasion of apoptosis, enhanced DNA repair and enhanced migration. The effects of ß-catenin silencing correlated with its putative role in chemo-resistance and DNA damage response. CONCLUSION: From our results we conclude that ß-catenin may contribute to HNSCC therapy resistance and disease relapse. As such, ß-catenin may be explored as a therapeutic target along with conventional therapeutics.

Clinico-Pathological Correlation of ß-Catenin and Telomere Dysfunction in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: Tumorigenesis is a complex process of accumulated alteration in function of multiple genes and pathways. Wnt signalling pathway is involved in various differentiation events during embryonic development and is conserved in various species. OBJECTIVE: A multicentre collaborative initiative is undertaken to study the occurrence, prognosis and molecular mechanism of HNSCC (Head and Neck Squamous Cell Carcinoma) which is highly prevalent in eastern parts of India. From a large cohort of HNSCC tissue repository, 67 cases were selected for multi-parametric investigation. RESULTS: 67 cases showed stable ß-catenin expression. We have seen correlation, if any, of the transcription factor - ß-catenin, telomere maintenance and shelterin complex proteins - TRF2, Rap1 and hTert with respect to tumor differentiation and telomere dysfunction. Immunohistochemistry of ß-catenin protein showed stable and high expression in tumor when compared to stroma. MDSCC (Moderately Differentiated Squamous cell carcinoma) cases expressed nuclear expression of ß-catenin in invasive fronts and showed increased genomic instability. Higher frequency of Anaphase bridges was observed ranging from <3% in normal cut margin to 13% in WDSCC (Well differentiated squamous cell carcinoma) and 18% in MDSCC (Moderately differentiated Squamous cell carcinoma). There was significant decrease in telomere length in MDSCC (<4) when compared to the normal cut margin samples (<7). Quantitative Real Time-PCR confirmed a significant correlationship between stable ß-catenin expression and poor clinical and pathological outcome. CONCLUSION: The Stabilisation and accumulation of ß-catenin was significant and correlated well with de-differentiation process as well as prognosis and therapy outcome of the patients in the cohort. Expression status of molecular markers such as ß-catenin, hTert, TRF2 and RAP1 correlate significantly with the process of tumorigenesis and prognosis and may play a role in therapeutic management of Head and neck patients.