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Year : 2014  |  Volume : 6  |  Issue : 2  |  Page : 99-103

PIK3CB and K-ras in oral squamous Cell carcinoma. A possible cross-talk!

1 Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, United Arab Emirates
2 Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, Iraq

Date of Web Publication16-Oct-2014

Correspondence Address:
Natheer H Al-Rawi
Department of Oral and Craniofacial Health Sciences, University of Sharjah, Sharjah
United Arab Emirates
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-8844.143049

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Background: PIK3 and K-ras are signal transducing proteins involved and mediating many responses related to cell cycle growth regulation. Until date, there has been only limited evidence about the expression of K-ras and PKI3CB in oral squamous cell carcinoma (OSCC). AIMS : This study aimed to examine both proteins in OSCC and their relation to clinic- pathological findings. Setting and Design: A total of 31 formalin-fixed paraffin-embedded specimens of OSCC were selected in this study. PIK3CB and K-ras expressions were detected using standard immunohistochemical techniques. Materials and Methods: PIK3CB and k-ras immune reactivity was semi-quantitatively evaluated in at least five representative fields at 400X magnification and recorded as percentage of PIK3CB and k-ras positive tumor cells over the total number of cells examined in the same area. Results and Conclusion: All examined specimens of OSCC were positive for monoclonal antibodies directed against PIK3CB and K-ras proteins especially at advanced stage of the disease. No significant relation was observed between the tested proteins and the clinic-pathological findings of OSCC; however a highly significant direct relationship was observed between K-ras and PIK3CB. This lead to conclusion that both K-ras and PIK3CB signaling pathway were activated in the advanced stage of OSCC, and possibly a cross-talk between them. This could make these mutant proteins a potential target for an effective molecular therapy.

Keywords: K-ras, PIK3CB, signal transduction, SCC

How to cite this article:
Al-Rawi NH, Merza MS, Ghazi AM. PIK3CB and K-ras in oral squamous Cell carcinoma. A possible cross-talk! . J Orofac Sci 2014;6:99-103

How to cite this URL:
Al-Rawi NH, Merza MS, Ghazi AM. PIK3CB and K-ras in oral squamous Cell carcinoma. A possible cross-talk! . J Orofac Sci [serial online] 2014 [cited 2023 Feb 1];6:99-103. Available from:

  Introduction Top

Oral cancer is one of the worst forms of cancer in terms of associated mortality and morbidity. Although significant progress has been made in early detection, diagnosis and treatment, the 5-year survival rates for patients with oral cancer have not improved in the past 30 years and have remain around 50%. [1] Understanding the molecular mechanisms involved in initiation and progression to malignancy of oral cancer will help improve the prognosis of the disease and develop a novel therapeutic strategies. Several pathways of signal transduction have been identified. Ras genes are the most frequently mutated oncogenes in human cancer. [2] The activating mutation in K-ras and H-ras has been reported to be overexpressed in oral cancer. [3] Amplification of wild type K-ras in oral cancer results in an overactive mitogenic signal, which in turn, result in an increased proliferative and/or survival response that contributes to the etiology of head and neck squamous cell carcinomas (HNSCC). [4] The phosphatidyl inositol 3-kinase (PI3K) signaling pathway is a crucial regulator of many normal cellular processes, such as cell growth, proliferation, motility and apoptosis, it is deregulated in a wide range of human cancers by gain-or-loss of function of several components of this pathway, including phosphatase and tensin homolog (PTEN), AKT and PIK3CA. [5],[6] The PI3K family encompasses lipid kinases that convert phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3, 4, 5-trisphosphate, which in turn initiates a signaling cascade that promotes cell growth and survival. [7] Recently, amplification and mutations of PIK3CA were reported in HNSCC. [8],[9],[10],[11]

However, no large scale analysis of PIK3CB expression and their clinico-pathological significance have ever been performed in oral squamous cell carcinoma (OSCC). Therefore, in the present study, human OSCC samples were analyzed for the expression profiles of K-ras and PIK3CB as well as to correlate their expression with clinico-pathological characteristic of the disease.

  Materials and methods Top

Thirty- one formalin-fixed paraffin- embedded tissue blocks of OSCC were used in this study. All hematoxylin and eosin (H&E) stained tissue sections were reviewed, and the best sections from primary tumor of each specimen were selected. Other 10 samples of histologically normal oral mucosa were also included and served as study control group. The study protocol was approved by the Institutional Review Board of the College of Dentistry, University of Baghdad-Iraq. All the 41 blocks were cut at 5μM thickness and mounted on poly-L-lysine coated slides. The sections were deparaffinized and stained by the standard immunohistochemical technique. Antigen recovery was conducted with citrate buffer (10 mmol/L in a microwave/800W) for 7 minutes. The reagents were used in a peroxidase-based system to identify antigen-antibody conjugates. The sections were incubated with primary antibodies against K-ras (1/100 in 0.025 triton X-100 TBS mouse monoclonal anti-K-ras antibody; Abacam ab5539). Other sections were incubated with primary antibody against PI3KCB (mouse monoclonal anti-PI3 kinase P110 beta antibody in 1/200 dilution; Abacam ab55593). The selected sections were then incubated overnight at room temperature after blocking endogenous peroxidase activity with 3% hydrogen peroxide. Negative control slides were obtained by omitting the primary antibodies. The slides were then washed in phosphate buffer saline (PBS) for five minutes. Then a biotinylated goat-anti-mouse IgG was applied and incubated for 10 minutes at room temperature. The immune staining procedure was performed using a labeled streptavidin-biotin system (LSAB system-HRP, Dakocytomation, Carpentaria, California, USA). Mayer's hematoxylin counterstain was then applied to all sections for 2 min, washed for another 2 min, dehydrated in a graduated alcohol concentration, and then mounted with DPX under a cover slip. The stained regions of interest were viewed at 400 X magnifications and scored. Staining of K-ras and PI3KCB was semi quantitatively in at least five fields. A score of 0-4 was used where grade 0 indicates (negatively stained cells), 1+ (5-20% positively stained cells), 2+ (20-40% positively stained cells), 3+ (40-80% positively stained cells), and 4+ (>70% positively stained cells). [12] Each specimen was scored individually. One observer scored all cases, which were rechecked randomly by the same investigator after a period of time. Statistical analysis of the immune scores was performed using SPSS version 18 (Chicago, IL).

  Results Top

Thirty one specimens of OSCC were selected in this study, of which 23 specimens (74.2%) were removed from patients aged more than 50 years, and the rest of the specimens (8 specimens 15.8%) were removed from patients aged less than 50 years. About two third (64.5%) of the cases were taken from male patients. The lips was the most common site (9 cases; 29%), followed by tongue, floor of the mouth and the mandible. Most cases (25 specimens) were histologically confirmed as well differentiated carcinoma (80.65%), 11 of them were at stage I and the rest were at stage IV. Four cases were moderately differentiated and only two cases were of poorly differentiated carcinoma. Nearly half of cases were at stage IV (14 case; 45.16%) [Table 1].
Table 1: K-ras and PIK3CB expression in OSCC

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K-ras and PI3KCB were expressed in more than 70% of the carcinoma cells (score 4) in nearly half of the cases (51.6% and 45.16%, respectively) followed by score3 (25.8% and 35.48%) respectively as seen in [Table 1].

The expression was in the form of brown stain of the cell membrane and the cytoplasm of tumor cells. Non-significant statistical differences in the K- ras and PI3KCBexpression were observed on age and gender based comparison. Well differentiated carcinoma in patients at stage IV had the highest K-ras and PI3KCBscore [Figure 1] and [Figure 2]. Highly significant statistical correlation was observed between the K-ras and PI3KCB expressions (P < 0.005) [Table 1].
Figure 1: Positive expression of K-ras in malignant cells of well differentiated OSCC (×400)

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Figure 2: Positive expression of PI3Kcb malignant cells of well differentiated OSCC (×400)

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A non-significant statistical difference in the expression of K-ras and PI3KCB among different histological grades [Table 2] and [Table 3] [Figure 3], [Figure 4], [Figure 5], [Figure 6] as well as among different clinical stages was observed [Table 4] and [Table 5]. It was demonstrated that cancer in advanced stages is usually well differentiated and expressed K-ras and PI3KCB more than as compared to that in early stages, albeit not reaching significant statistical differences.
Table 2: Correlation between K-ras and the tumor grade of OSCC

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Table 3: Correlation between PIK3CB and the tumor grade of OSCC

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Table 4: Correlation between K-ras and the tumor stage of OSCC

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Table 5: Correlation between PIK3CB and the tumor stage of OSCC

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Figure 3: Positive expression of K-ras in malignant cells of moderately differentiated OSCC (×400)

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Figure 4: Positive expression of PIK3cb in malignant cells of moderately differentiated OSCC (×400)

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Figure 5: Positive expression of K-ras in malignant cells of poorly differentiated OSCC (×400)

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Figure 6: Positive expression of PIK3cb in malignant cells of poorly differentiated OSCC (×400)

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  Discussion Top

RAS and RAS-related proteins are often deregulated in cancer leading to increased invasion, metastasis and decreased apoptosis. RAS activates several pathways, of which Mitogen-activated protein (MAP) kinases cascades has been well studied. This cascade transmits signals downstream and results in the transcription of genes involved in cell growth and division. [13] Activating mutations in K-ras and H-ras have been reported in human SCC, primarily in those caused by exposure of chemical carcinogens. [2] PIK3 amplification and mutations were also reported in HNSCC. [8],[9] The phosphatidyl-inositol-3-kinase is a heterodimer lipid kinase that plays a pivotal role in regulation of various cellular signaling pathways which control cell proliferation, survival, growth, motility, cell adhesion, differentiation, cytoskeletal rearrangement and apoptosis. [5] PIK3 family is divided into 3 different classes: I, II & III. The class IA can be activated by being recruited to the cell membrane via growth factor receptor (GFR) tyrosine kinase, such as EGFR and insulin receptors. [14] Growth factors activates receptor tyrosine kinases (RTKs), which then activate two key signal-transduction components including the small GTPase Ras and the lipid kinase PI3. Active Ras could also directly activates PIK3. [15] The PIK3CA, which codes for the P110 α catalytic subunit of PIK3 has been reported to be amplified in human cancer including HNSCC.[16] In the present investigation, the IHC expression of both K-ras and PIK3CB were detected in all cases of OSCC, regardless of the age, gender, site or the clinical stage of the tumor. These findings were in accordance to the results of previous study. [17] The highest score of expression was noted in well- differentiated SCC cells. The expression was more pronounced as the stage of advanced tumor. A highly significant positive correlation was observed between the expressions of K-ras and PIK3CB in the present investigation. Estilo et al., [16] suggested that PIK3-AKT as well as K-ras pathways are important signaling pathways that contribute to oral cancer development, especially at an advanced stage due to the cumulative genomic mutation. Also the activation of PIK3CB due to mutation, rather than amplification, may strongly contribute to deregulation of PIK3 signaling pathway in the advanced progression, especially in stage IV of OSCC. TheRAS and PI3K pathway mutations were found to be mutually exclusive in breast cancers, and a significant fraction (22%) of colorectal cancers has genetic lesions in both pathways. [18],[19] The molecular significance and therapeutic implications, however, of co-occurring mutations in the PI3K and RAS pathway are presently unclear. Hoa et al., [4] suggested that K-ras expression enhanced both the growth and survival of the primary keratinocytes under their experimental condition. This is in accordance with the findings of the present investigation, where K-ras was expressed in the cytoplasm of more than 70% of the keratinocyte tumor cells. Recent in vitro and in vivo studies indicated that increased expression levels of mutated Ras genes could potentially play an important role in the development and progression of OSCC. [20],[21] Yabrought et al., [21] found that an increased expression levels of K-ras due to amplification resulted in a significant proliferative response and increased cell survival and, hence, tumor progression. Estilo et al., [16] and Woenckhaus et al., [8] found that gene amplification and over-expression of PIK3CA are associated with transition of invasive cancer, which is in accordance with the results of the present study. Wee et al., [22] showed that inactivation of either the RAS and MEK or PI3K pathway leads to partial tumor growth inhibition and that the targeted inhibition of both the pathways is required to achieve tumor stasis. They suggested that the PI3K pathway activation strongly influences the sensitivity of RAS mutant cells to MEK inhibitors. Activating mutations in PIK3CA reduce the sensitivity to MEK inhibition, whereas PTEN mutations probably cause complete resistance. Recent studies indicate that PIK3CB, rather than PIK3CA, is the major PI3K isoform that drive the proliferation of PTEN mutant cancer cell lines. [7],[23] The strong correlation between K-ras and PIK3CB expression in the advanced stage of OSCC in the present study is in accordance with the report of Heinemann et al., [24] who suggested that PI3K is activated by recruitment to the cell surface by activated receptor tyrosine kinases (like EGFR)in colorectal cancer, as well as by binding to activated RAS. The mutation of both K-ras and PIK3CBin addition to EGFR mutation could be the main mechanism by which OSCC progression occurs. Possibly due to the extensive cross-talk between them. Further studies are needed to explore this cross talk. Further studies are required to unveil this cross talk is indicated specially on genomic level. A combination therapy with PI3K and MEK inhibitors can be further tested to stop oral cancer progression.

  Conclusion Top

Both K-ras and PIK3CB signaling pathway were activated in the advanced stage of OSCC, which makes these mutant proteins a potential target to achieve an effective molecular therapy.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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