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ORIGINAL ARTICLE
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Dysregulated claudin expression significantly effect breast cancer disease progression


1 Department of Biosciences, COMSATS University, Islamabad, Pakistan
2 Department of Molecular Biology, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan
3 Department of Surgery, Holy Family Hospital, Rawalpindi, Pakistan

Date of Submission07-Apr-2020
Date of Decision01-Jun-2020
Date of Acceptance10-Sep-2020
Date of Web Publication05-Aug-2021

Correspondence Address:
Muhammad Faraz Arshad Malik,
Department of Biosciences, COMSATS University Islamabad, Park, Road, Islamabad
Pakistan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_427_20

 > Abstract 


Background: In this study, the role of claudins in cancer progression was explored among breast cancer-affected women.
Methodology: Two cohorts (discovery and validated) of breast cancer-affected women were used. In discovery cohort, 90 freshly excised breast tumor tissues along with adjacent cancer free specimens were collected at the time of surgery. These specimens were processed for RNA isolation and complementary DNA synthesis. After designing primers for claudin 3, claudin 4, and claudin 7, these sequences were synthesized from Macrogen, Korea. Claudin expression in respective tumors and controls was assessed using quantitative reverse transcription polymerase chain reaction. Any probable correlation of these molecules with various clinicopathological parameters was explored. For validation, a publicly available dataset of 2088 breast cancer patients was accessed. Claudin expression of these patients was analyzed for given clinical parameters and compared with earlier findings of discovery cohort.
Results: Discovery cohort comprised 17% luminal A, 63% luminal B, 8% human epidermal growth factor receptor 2 enrich, and 12% triple-negative breast cancer tumor. High claudin 3 expression was significantly correlated with tumor size >2 cm and menopausal status. Claudin 7 expression was upregulated among poorly differentiated tumor patients. Both claudins 3/4 showed significant correlation with tumor grade, stage, size, and metastasis. Claudin-low subtype was also found in 18% of the cohort.
Conclusion: Claudins impart a significant role in cell differentiation and disease progression. Hence, claudin cluster can be ascertained as the disease biomarkers for breast cancer.

Keywords: Breast cancer, claudins, cohort



How to cite this URL:
Fatima Z, Riaz SK, Khan JS, Haq F, Malik MF. Dysregulated claudin expression significantly effect breast cancer disease progression. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=323172




 > Introduction Top


Breast cancer is ranked first among the most commonly encountered cancers affecting women in Pakistan. Being a solid tumor, the integrity of tight junctions in mediating cell polarity and interaction with surrounding microenvironment is vital. Claudins and occuldins are core proteins responsible for the formation of these junctions. Claudin family comprises 27 integral membrane protein members, with a largely uniform structural similarity. Majority of the claudins family members comprise of two extracellular loop structures followed by four transmembrane protein domains joined to PDZ domain present inside the cytoplasm. PDZ domain present at carboxyterminal region is interacts with numerous cellular proteins including ZO-1, ZO-2, ZO-3, symplekin, cingulin, PATJ, MAGI-1 (MAGUK), AF-6, and PALS.[1] Claudins act as adaptors by directly or indirectly interacting with regulatory proteins, cytoskeletal molecules, and transcription factors.[2] These proteins are also associated with numerous signaling pathways, such as JAK-STAT3, WNT, and notch signaling, responsible for cancer progression.[3],[4] Interestingly, claudins expression may also be altered by epigenetic regulation including histone modifications and DNA methylation.[5] Epithelial–mesenchymal transition (EMT) markers including Snail also act as transcriptional repressor for claudins 1, 3, 4, and 7.[6] Claudin expression significantly varies depending on organ involved and type of tumor.[7] High claudin 3 and 4 expression was observed in ovarian cancers, in contrast reduced claudin 4 was noted in pancreatic cancer.[8] Similarly, claudin 7 differentiates between hepatocellular carcinoma and cholangiocarcinoma.[9] Overexpression of claudin 4 in gastric cancer and of claudin 3 in pancreatic cancer, ovarian cancer, breast cancer, and endometrial cancer has been observed.[10],[11],[12],[13] Interestingly, downregulation of claudin 4 in esophageal and pancreatic cancer is associated with poor prognosis, while lower expression of claudin 7 in oral cancer is associated with better prognosis.[14],[15]

Reduced claudin 7 expression decreases with several lymph node involvement and metastasis, while in another study, increased expression of claudin 7 in metastasis was observed.[16],[17] These variations require a holistic approach to analyze the cluster of claudins against breast cancer. In addition, tumor heterogeneity significantly influences disease outcome, treatment response, and chance of relapse. Based on molecular biomarkers such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki67, breast tumors are categorized in four different molecular subtypes. These as termed as luminal A, luminal B, HER-2 enriched, and triple-negative breast cancer (TNBC). A fifth subtype which is recently identified is termed as claudin-low breast cancer. This subtype typically possesses highly distinctive histologic grade and EMT features with low/or no expression of cell junction proteins. This subtype poses an immense challenge based on the immune cell infiltration, interferon gamma activation, and poor response to therapy.[18] It proposes a new challenge for cancer research, due to its rarity and intricate features.

In the present study, claudin 3, 4, and 7 expression in respective breast cancer patients was estimated. Expression profiles of claudins were correlated with clinical parameters to investigate their clinical relevance. In addition, combined analysis of claudin 3 and 4 was also assessed to gain useful insight about their role in cancer progression.


 > Methodology Top


Two study cohorts were ascertained for the evaluation of claudin expression in breast cancer. In discovery cohort, Pakistani breast cancer patients from collaborating hospitals (Holy Family Hospital and Pakistan Institute of Medical Sciences) were enrolled. The study was conducted with prior approval of Ethical Review Committee set forth by the Institutional Biosafety and Bioregulation Committees of both CUI and respective hospitals. In validation cohort, in silico assessment of claudin expression was independently cross-checked in 2088 breast cancer patients.

Discovery cohort

In the cohort, 90 specimens were collected at the time of surgery along with their adjacent normal controls. After immersing in RNAlater® (Sigma-Aldrich, Darmstadt, Germany), these specimens were stored at −80°C. Additional information regarding age, menopausal status, area affected, tumor stage, grade, and tumor sizes was also collected in subsequent follow-up from clinicopathological reports.

RNA extraction and complementary DNA synthesis

RNA was isolated by using TRIzol® (Invitrogen, California, USA) method from both tumor and control tissues.[19] Complementary DNA (cDNA) was synthesized using Revert Aid First Strand cDNA Synthesis Kit (Solis BioDyne) as per the manufacturer's instruction.

Gene expression analysis of breast cancer cohort

Primers were synthesized from Macrogen (Korea) and retrieved in the lyophilized form. Primer sequences of these genes are CLDN3 (forward: 5'-CTGCTCTGCTGCTCGTGTCC-3'; reverse: 3'-TTAGACGTAGTCCTTGCGGTCGTAG-5'), CLDN4 (forward: 5'-GGCTGCTTTGCTGCAATGTC-3'; reverse: 3'-GAGCCGTGGCACCTTACACG-5'), CLDN7 (forward: 5'-CCACTCGAGCCCTAATGGTG-3'; reverse: 3'-GGTACCCAGCCTTGCTCTCA-5'), ER (forward: 5'-CCACCAACCAGTGCACCATT-3'; (reverse: 3'-GGTCTTTTCGTATCCCACCTTTC-5'), PR (forward: 5'-ATTACCAGTGTT CCCGTCTTC; reverse: 3'-CCTGTACTTCCTCCAGCATAA-5'), HER-2 (forward: 5'-TTGAGTCCATGCCCAATCC-3'; reverse: 3'-GTGTTCCATCCTCTGCTGTC-5'), Ki67 (forward: 5'-GCCTTGGTCTCTTGGGAATAC-3'; reverse: 3'-GGAGATTAGGAGCCAGTTTGAG-5') and GAPDH (forward: 5'-CAAGGTCATCCATGACAACTTT-3'; reverse: 5'-GTCCACCACCCTGTTGACAACTTG-3'). GAPDH was used as the internal reference control. Polymerase chain reaction conditions included initial denaturation at 94°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and concluding at 4°C.

Validation cohort

In silico data collection

To validate discovery cohort findings, the dataset of breast cancer-affected patients obtained from array express (ID: E-MTAB-6703) with a cohort size of 2088 patients was used. Any probable correlation of aforementioned genes with different clinicopathological parameters such as tumor stages, grade, size, metastasis, node involvement, patient age, and menopausal status was assessed.

Statistical analysis

To find the correlation between claudin expression, their molecular subtypes, and clinical parameters, Chi-square test, Wilcoxon signed-rank test, and Mann–Whitney test were used.


 > Results Top


Claudin expression profiling in the discovery cohort

At mRNA level, claudin 3 expression was found high 40% (n = 36) of patients. Similarly, for claudin 4 and claudin 7, overexpression in 52% (n = 47) and 45% (n = 50) cases was observed, respectively. Expression of ER, PR, HER-2, and Ki67 was also evaluated in the cohort. Claudin 3 showed positive correlation with PR-negative and Ki67-positive samples [Figure 1] and [Table 1].
Figure 1: Heatmap showing expressional variation of claudin and molecular biomarkers in discovery cohort

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Table 1: Clinicopathological categorization of discovery cohort

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Effect of claudins with tumor grade and stage in discovery cohort

Data demonstrated that the majority of these samples belong to Grade II (51/90) and Grade III (34/90) in the discovery cohort. Claudin 3 was found to be significantly high among poorly differentiated tumors. A significant association of claudins with grade and stage was noted [Table 1].

Effect of claudins with tumor size, nodal involvement and metastasis

Highest number of patients was associated to large tumor size (82%), positive nodal involvement (82%), and lacking metastasis (93%). Claudins 3 expression was significantly high in tumors >2 cm size [Table 1].

Correlation with age and menopausal status

Patients' age range in the discovery cohort was between 23 and 94 years. Women aged >48 years showed significantly higher expression of claudin 3 and claudin 4. On the contrary, premenopausal breast cancer-affected women showed significantly upregulated expression of claudin 3 [Table 1].

Molecular subtyping of the cohort

Cohort was divided into five molecular subtypes, including luminal A (17%), luminal B (63%), HER-2 enriched (8%), and TNBC (12%). Sixteen (18%) out of 90 samples did not show any expression for all three claudins. These are designated as claudin-low subtype. Results showed that the presence of claudin 3 and claudin 4 in luminal cancer is higher. Majority of cases were from luminal B subtype (57/90). Luminal A was the second most common type following TNBC and HER-2 enriched. All claudins show different expression profile according to the subtypes of breast cancer. All three genes are upregulated in TNBC samples.

Expression analysis of claudins in validation cohort

We extracted gene expression data for all three claudins (n = 2088) to validate our findings from in vitro study. Results revealed that claudin 3 and claudin 4 were significantly associated with late tumor grade. Claudin 3 also showed significant association with advanced tumor stage as well. In addition, claudin 4 showed association with ER-negative, PR-negative, and HER-2-positive samples.

Combine expression of claudin 3/claudin 4

Based on similar sequences and functional properties, both claudin 3 and claudin 4 coexist on cell membranes. After combining the expression levels of both genes, we found that high expression of claudin 3 + 4 is associated with tumor size, advanced tumor grades, stages, and metastasis. However, no association with lymph node involvement was found. In case of molecular biomarkers, positive association was seen for PR-negative samples. However, in in silico data, significant correlation was found for ER-negative and PR-negative samples. It also showed higher positivity with late tumor stage and grade [Table 2].
Table 2: Association of claudins with molecular biomarkers

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 > Discussion Top


Dysregulation of claudins has widely been reported in different cancers.[19] Expression of claudins was not only tissue specific but also unique in certain subtypes. In a recent study, claudin 7 expression in TNBC patients exhibited marked overexpression in poorly differentiated tumor specimens.[20] Low expression of claudin also significantly affects tight junction strength and metastasis of breast cancer cells. Hence, low claudins not only affect tumor progression but also reduce cell adhesion.[21]

This study was designed to evaluate the expression profile of claudin 3, claudin 4, and claudin 7 in breast cancer cohort of Pakistan. Selection of claudin 3, claudin 4, and claudin 7 was based on the evidence describing their low expression in claudin-low subtype. Claudin 7 did not show any significant association with majority of markers. In our study, interestingly, positive association of claudin 7 with tumor grade was established. Moreover, claudin 3 showed association with tumor stage. However, when combined analysis of phylogenetically related claudin 3 and claudin 4 was done, higher level of association with poor differentiation of tumor and late stages was observed in both cohorts. In vitro dataset, positive relation of claudin 3/4 was found for samples having metastasis. These results are also consistent with previous observations.[22]

Many studies have shown that level of androgen is involved in the formation and proliferation of breast tumor. Absence of androgen is associated with large tumor size, higher grade, and nodes involvement.[23] This concept supports expression profiling of claudin 3 in the present study as it is significantly higher in tumor >5 cm. Claudin regulation by several EMT transcriptional factors including Sip1, Zeb1, and vimentin was also reported. Extremely tight connection between claudin 3, Sip1, and EMT-mediated regulation of claudins was noted.[24] Sip1 was previously reported as a prognostic factor involved in metastasis.[25] Interestingly, claudin 3 overexpression was significantly regulating malignancy potential in other cancer of epithelial origin.[26]

Claudin-low subtype was first identified (2007) for breast cancer and characterized by lower expression of tight junction genes, including claudin 3, 4, and 7.[27] Claudin-low tumor exhibit less E-cadherin and high vimentin, Snail, and ZEB transcription factors.[28] In the present study, only 16 samples showed no expression for all three claudins. A recent study on claudin 7 expression in TNBC patients showed marked overexpression in poorly differentiated tumor specimens.[20] Another study conducted on invasive breast cancer patients showed a strong correlation of claudins with various molecular subtypes, such as both claudin 7 and claudin 8 associated with luminal tumors (ER+), CLDN1, and CLDN4 observed in basal-like tumors.[29] Interestingly, low claudin expression, especially claudin 3, 4, and 7 and E-cadherin, was significantly related in the same study.[29] According to a study, expression profiling of claudin 3 and claudin 4 was high among highly proliferative tumors stained with Ki67.[30] Data were also distributed according to the subtypes of breast cancer, and similar findings were observed in the present study stating that the presence of claudin 3 and 4 in luminal cancer is higher. Majority of cases were from luminal B subtype including both HER-2-positive and HER-2-negative samples. Luminal A was the second most common type followed by TNBC and HER-2 enriched. Our findings of claudin-low are justified by the fact that majority of patients in the study belong to luminal B subtype. Expression of Ki67 increases with the level of claudin 3 and claudin 4. Almost similar findings were observed in the present study as claudin 3 showed positive association with high level of Ki67. They also indicated Ki67 as a key sign for proliferation.


 > Conclusion Top


Both claudin 3 and 4 overexpression is significantly related to cell differentiation. Similarly, CLDN3 and CLDN4 also influence tumor staging. Hence, screening of CLDN3 and CLDN4 on the large cohort data is recommended.

Ethics approval and consent to participate

The study proceeded with prior approval from biosafety and ethical committees of COMSATS University Islamabad, Pakistan Institute of Medical Sciences, and Holy Family Hospital. Informed consents from the participants were collected after a thorough briefing of the proposed research.

Availability of data

Data regarding individual staining results can be furnished upon request.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

This study was supported by grants from Higher Education Commission of Pakistan under project ID 2989 and COMSATS University Islamabad, Pakistan.

Conflicts of interest

There are no conflicts of interest.



 
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