Role of Annexin 7 (ANXA7) as a tumor suppressor and a regulator of drug resistance in thyroid cancer

doi:10.21203/rs.3.rs-4360239/v1

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Role of Annexin 7 (ANXA7) as a tumor suppressor and a regulator of drug resistance in thyroid cancer

https://doi.org/10.21203/rs.3.rs-4360239/v1

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Thyroid cancer ranks as the predominant endocrine malignancy in the United States, boasting a generally favorable prognosis. However, a subset of patients faces a grim outlook due to the emergence of drug resistance. The progression and aggressiveness of thyroid cancer have been linked to the differential expression and mutation of key genes such as BRAF, Met, and p53, with the V600E mutation in BRAF present in over 60% of cases. ANXA7, a versatile protein with tumor-suppressive properties observed across various cancers, warrants attention concerning its role in thyroid cancer. Our investigation delved into the interplay between ANXA7 expression, BRAF mutation, and their impact on disease progression, aggressiveness, and drug response.

High-throughput RNA-seq and protein array analyses revealed diminished ANXA7 expression in thyroid cancer, particularly in cell lines harboring the BRAF mutation. Treatment of thyroid cancer cells with BRAF and MEK inhibitors led to upregulated ANXA7 expression, decreased Ph-ERK levels, and increased apoptotic markers. Notably, our findings unveiled the cyclin-dependent kinase inhibitor p21 as a novel regulator of BRAF-mediated chemoresistance. Combining drugs to elevate both p21 and ANXA7 levels synergistically enhanced apoptotic signaling. These discoveries shed light on a novel pathway implicated in thyroid malignancy and drug resistance, involving the ANXA7/p21/BRAF/MAPK axis. Our study marks the first elucidation of this pathway, offering promising insights into overcoming resistance to BRAF or MAPK-targeting drugs into treatment of thyroid cancer or even BRAF mutation mediated melanoma. Future translational endeavors, leveraging high-throughput functional screenings, are imperative for developing innovative ANXA7-based therapeutic strategies tailored to thyroid cancer.

Biological sciences/Cancer

Health sciences/Oncology

ANXA7

p21

BRAF E600V mutation

chemoresistance

Nutlin-3A

Thyroid cancer (TC) is one of the most common malignancies in the United States. This disease also has one of the highest survival rates with 98.2% of people surviving in 5 years ¹. Although most TC can be treated successfully with surgery, radioiodine ablation, or thyroid suppression therapy, tumor recurrence occurs in 5–20% of patients ². If distant tumor recurrences occur, the 10-year survival rate of TC is reduced to approximately 40%. The development of molecular determinants of disease recurrence has the potential to improve the clinical management of patients with TC by assisting in risk stratification and combined uses of more aggressive treatments with patients who are at higher risk of recurrence ^3–6.

The BRAF V600E mutation is the most common genetic mutation detected in patients with TC ⁷. A large multicenter retrospective study by Xing et al. ⁸ found that in an unadjusted analysis, the presence of the BRAF V600E mutation was significantly associated with TC-related mortality. Another study by Kong et al., clearly showed the importance of the BRAF V600E mutation in making the TC drug-dependent ⁹. The evolution of chemoresistance in patients undergoing treatment is a risk factor for more aggressive forms of the disease or worse outcomes ⁹.

Different cell compartments are involved in the mechanism of chemoresistance, and multiple mechanisms can be activated by single cells at different times of the cancer progression. For example, the alteration of drug metabolism, derangement of intracellular pathways’ signaling, cross-talk between different membrane receptors, and modification of apoptotic signaling and interference with cell replication are all mechanisms that the cell uses to overcome the effect of pharmacological compounds ¹⁰. Additionally, activation of tyrosine kinase receptor systems specific signaling cascades leading to the activation of key pro-survival pathways such as PI3K/Akt/Ras/ Raf/MEK/Erk, and STAT are implicated ^{11,12 13}. BRAF is a serine-threonine protein kinase, which plays a critical role in aggressive melanomas ¹⁴. But the role of BRAF in less aggressive cancers, such as TC, is less known. We focused on the most well-known mutation BRAF V600E associated with drug resistance in this study on a TC model. BRAF V600E and aggressive clinic-pathological behaviors are highly correlated, as described by Xing et al. ¹⁵. Thus, BRAF V600E does have a significant association with mortality, which likely occurs through promoting aggressive tumor behaviors.

The ANXA7-GTPase is a tumor suppressor, frequently inactivated by genomic alterations at 10q21 ¹⁶. In the last few years, considerable amounts of data have accumulated describing the inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase ^{16–23 24}. ANXA7-GTPase contains a calcium-binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment ^{12,25,26 16}. Tumor suppressor function of the calcium/phospholipid-binding Annexin-A7 (ANXA7) has been shown in ANXA7-deficient mice and validated in human cancers ¹⁷. In the androgen-resistant prostate cancer cells, ANXA7 and p53 showed similar cytotoxicity levels. However, in the androgen-sensitive LNCaP, ANXA7’s level greatly exceeded the p53-induced cytotoxicity ¹⁷.

In this study we examined the relationship between BRAF and ANXA7 in TC progression and chemoresistance. We focused on the BRAF V600E mutated strains, as they are known to increase drug resistance in vitro or in vivo when exposed to pharmacological agents ²⁷. We found that ANXA7 has a tumor suppressive role in thyroid cancer. Besides, we also found correlation between ANXA7 and p21, which are associated with BRAF-mutation mediated ERK/MEK activation. The p21 protein, also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a cyclin-dependent kinase inhibitor (CDKI) that is capable of inhibiting all cyclin/CDK complexes; It is also a downstream effector of p53 ²⁷. Hence, any drug that could increase the p21 activity will have great significance in controlling chemotherapy. However, p21-based drug therapies are hard to come by, as p21 is involved in a central mechanism that normal cells depend on to be differentiated and integrated into the specific tissue specialized in function. Here, we found the additive effect of BRAF and p21 inhibition, which could potentially produce a new chemotherapeutic approach and be beneficial to recurrent TC patients and extended to melanoma patients.

It is well-known that the BRAF mutation is associated with drug-resistance for several cancers including melanoma ^28,29. In this current study, we found that the upregulation of ANXA7 expression is an indicator of drug-sensitivity towards BRAF inhibitors. In addition, the combinatorial use of BRAF and p21 inhibitors are lethal to the drug resistant form of TC carrying BRAF V600E mutations. This has translational significance, as TC mutational analysis may dictate the need for early combined treatment with BRAF and p21 modulators to improve the outcomes in high-risk cases. The axis of ANXA7-BRAF-p21 correlation is a novel outcome of this current study. The upregulation of ANXA7 in combined therapy of BRAF and p21 modulators resulted in increased drug sensitivity, and hence this combination could be a novel tumor suppressive axis.

Expression of BRAF and ANXA7 in different tissues including thyroid glands.

In this current study, we analyzed the expression of BRAF and ANXA7 in different tissues, including the thyroid glands. We assessed the organ-wide expression of ANXA7 and BRAF RNA, as well as protein expression levels, by analyzing data from the Human Protein Atlas (www.proteinatlas.org) ³⁰. BRAF emerged as an important cellular protein with a significant expression level in almost all kinds of tissues, while the highest expression of ANXA7 in normal tissue was observed in the endocrine glands (thyroid gland, particularly in the parathyroid gland) (Fig. 1a-b). This finding may explain the biological function of Annexin A7 (ANXA7), which belongs to the annexin family of calcium-dependent phospholipid-binding proteins. Additionally, we analyzed mRNA data from tumor samples for these two markers (BRAF and ANXA7). The data indicated that the expression of ANXA7 is lower in cancer samples, whereas the expression level of BRAF remains constant (Fig. 1c-d, Supplementary data Fig. 1s). We also analyzed cell line data from the Cancer Cell Line Encyclopedia (CCLE) and from MCLP for these two proteins (Fig. 2 and supplementary data Fig. 1s). The protein expression data from cancer cell lines is consistent with the data from tumor samples. Specifically, the expression of ANXA7 is lowest within thyroid cancer cell lines compared to 19 other different cancer types, totaling over four hundred cancer cell lines (data analyzed from MD Anderson Cell Lines Project, https://tcpaportal.org/mclp/#/) ³¹.

To validate our findings, we performed wet lab experiments. We utilized four cell lines named FTC-133, MDA-T68, K1, and 8505C. Both K1 and 8505C have a BRAF V600E mutation, which was also verified with Western blot data using a specific antibody. The mutational status of KRAS, TP53, BRAF, PIK3CA and PTEN in these cell lines and in other thyroid cancer cell lines are also mentioned in the supplementary data Table 1s. The expression of ANXA7 in FTC-133 is very high, and the phospho-ERK expression is the lowest, while the inverse is true with the cell lines harboring BRAF V600E mutation (Fig. 2d-e). These results show that the inflammatory pathway is activated by the BRAF V600E mutation, which is confirmed by the expression of P-ERK in these two cell lines (Fig. 2d-e).

Table 1

Antibodies Used in Simple Western Analyses.
Antibody	Dilution	Company
ANXA7	1:50	BD Biosciences
B-RAF V600E	1:100	Bio SB Inc
ERK	1:100	Cell Signaling Technology
Ph-ERK	1:100	Cell Signaling Technology
Rb	1:100	Cell Signaling Technology
P-Rb	1:100	Cell Signaling Technology
Cyc D1	1:10	Santa Cruz Technology
Cleav. Caspase 3	1:50	Cell Signaling Technology
p21	1:50	BD Biosciences
β-Actin	1:200	Sigma
β-Tub	1:50	Upstate

The regulation of ANXA7 expression by BRAF mutation.

To further investigated the regulatory role of BRAF mutation on ANXA7 expression, we analyzed large volume data sets from the curated databases cBioportal and CCLE. In about 60% of the thyroid cancer cases, BRAF mutations are present (Fig. 3, and Fig. 1s, Table 1s for cell lines). The BRAF gene is a proto-oncogene. The highest level of BRAF mutation is through the V600E mutation (cBioPortal). When the mRNA expression levels were viewed, both BRAF and ANXA7 had mid-range levels in thyroid cancer cases (Cancer Cell Line Encyclopedia, CCLE). However, when looking at the protein expression levels of these genes through Reverse Phase Protein Array (RPPA), ANXA7 expression was lowest in thyroid cancer (MD Anderson Cell Line Project, Fig. 2). Since it is the BRAF mutation and not the expression that leads to cancer, we decided to look more closely at ANXA7.

Ph-ERK and MEK inhibitors induce ANXA7 expression by activating the apoptotic pathway.

The four cell lines, with MDA-T68, K1, 8505C, and FTC-133, were tested with antibodies with Simple Western analyses through the Wes machine. As indicated in Fig. 2, these cell lines were shown to have varying levels of expression with the ANXA7, Ph-ERK, and BRAF antibodies. They were tested with BRAF V600E to confirm that K1 and 8505C were the two with the expression of the BRAFV600E mutation. These two also had higher Ph-ERK levels of expression, in comparison to MDA-T68 that had low expression of Ph-ERK and FTC-133 that had no noticeable expression. When ANXA7 expression was normalized among the four cell lines, FTC-133 had the highest expression, followed by MDA-768, whereas K1 and 8505C had the lowest levels of ANXA7 expression (Fig. 2d-e). The inverse relationship between higher levels of BRAF V600E and ANXA7 can be seen when comparing these cell lines, indicating a tumor suppressive role in thyroid cancer that is inhibited with the aggressive and oncogenic BRAF V600E mutation.

Inhibition of ph-ERK by different inhibitors.

Since BRAF V600E mutation plays a significant role in regulating the MAP kinase/ERK’s signaling pathway, which affects cell division, differentiation, and secretion ³², we used different inhibitors to block ph-ERK activity. Specifically, the drugs SB590885, AZD6244, GDC-0879 (list of drugs and their structures are in Fig. 4 and Table 2) were used to test their effects on ANXA7 and Ph-ERK over a 24-hour and 48-hour period. These Simple Western assays were performed on both the K1 and 8505C cell lines that had the BRAF V600E mutation. As seen in Fig. 5, the cell lines were treated with each drug alone and in combination and tested with ANXA7, Ph-ERK, ERK, and Cleaved Caspase 3 antibodies against a control. As primarily seen in 8505C, as K1, Ph-ERK increased and ANXA7 decreased when compared to the control in the first 24-hour period. After 48-hour period, the Ph-ERK decreased with an increased ANXA7 expression with the drugs and mostly the drug combinations. This shift correlated with an increase in cleaved Caspase 3 (apoptotic marker) along with an increasing expression of ANXA7, more cells were dying in the 48-hour period. It is important to mention that the potential for the development of resistance in cancer cells (8505C) when treated with inhibitors of the BRAF or MAPK pathways. These pathways play critical roles in cell growth, proliferation, and survival, and their dysregulation is often associated with cancer development and progression. However, cancer cells can develop mechanisms to bypass or counteract the effects of targeted therapies, leading to treatment resistance. The presence of surviving cells following drug treatment suggests the potential emergence of a resistant population within the cell lines.

Table 2

Drugs Used as B-Raf and MEK Inhibitors.
Name	IC50	Pathway to Inhibit
SB590885	0.16 nM	B-RAF
AZD6244	14 nM	MEK
	10 nM	ERK ½ Phosphorylation
GDC-0879	63 nM	pERK/B-RAF
Nutlin-3A	90 nM	MDM2/p53
Bortezomib	0.6 nM	ERK/20S Proteasome

The increase in ANXA7 expression can induce apoptosis in thyroid cancer cells.

As mentioned earlier, ANXA7 expression is crucial for thyroid cancer. Both data from cell lines and patients' samples indicated that ANXA7 decreases during pathogenesis. It is also hypothesized that the downregulation of ANXA7 expression could serve as an early and stage-specific biomarker for thyroid cancer or other thyroid-related malignancies^33,34. Therefore, it is of paramount importance to identify a drug that could increase ANXA7 expression regardless of the mutation level of other corresponding genes. We analyzed data from the MD Anderson Cell Lines Project (MCLP) for the protein-drug relationship (Volcano graph, Fig. 6a). This analysis indicated that the drug Azacitidine (in the figures indicated as ‘Aza’) up-regulates ANXA7 and sensitizes the cells to apoptosis (Fig. 4 for the structure of the drug and Fig. 6 for its effect of drug on 8505C). The increase in ANXA7 in 8505C cells is observed in a dose-dependent manner with Azacitidine (Fig. 6b). These results suggest that the upregulation of ANXA7 expression has the potential to trigger apoptosis in thyroid cancer cells.

Induction of p21 through Nutlin-3A drug treatment.

Understanding the mechanisms driving drug resistance is of paramount importance for enhancing cancer treatment outcomes. Combination therapies, which target multiple pathways concurrently or integrate immunotherapy, are being explored to combat or delay resistance development and enhance treatment effectiveness. In our investigation, we aimed to identify additional pathways that could serve as potential targets for combination therapy to overcome resistance induced by inhibitors of the BRAF/MAPK axis.

Our previous study indicated that the ANXA7 is linked to RB/p21 axis and protects normal prostate cells and induces distinct patterns of RB-associated cytotoxicity in androgen-sensitive and -resistant prostate cancer cells³⁵. In this study, we focused on ^33,36,37p21, also known as cyclin-dependent kinase inhibitor 1 (CDKN1A), a pivotal protein regulating the cell cycle. Acting as a tumor suppressor, p21 inhibits cyclin-dependent kinases (CDKs), pivotal enzymes driving cell cycle progression. Our study revealed that p21 expression levels were notably elevated in thyroid cancer cells, with distinctions observed based on the presence of the various BRAF V600E mutation (Fig. 7a-b). Further analysis utilizing MCLP data elucidated the relationship between drugs and proteins, showcasing Nutlin's ability to increase p21 expression and sensitize cancer cells to apoptosis (Fig. 7c). This observation aligns with existing literature demonstrating Nutlin's capability to induce p21 across various systems ^38–40. We are utilizing Nutlin 3A over Nutlin 3 primarily due to its superior potency and efficacy. Nutlin 3A is an enantiomeric isoform that has been demonstrated to be highly potent in inhibiting the interaction between p53 and MDM2. This interaction inhibition is crucial for restoring p53 activity, which is a key tumor-suppressor mechanism.

Employing Nutlin 3A to upregulate p21, our results demonstrated successful modulation of p21 expression within our experimental systems. Notably, we uncovered a synergistic effect of Nutlin 3A when combined with ph-ERK inhibitors (particularly Bortezomib), leading to augmented tumor cell death and complete suppression of colonies previously resistant to ph-ERK inhibitors alone (Fig. 7d-e). This novel discovery highlights Nutlin 3A as a potential pharmacological target for resistant thyroid cancers with poorer prognoses. Co-treatment with Nutlin 3A not only normalized ERK expression but also restored retinoblastoma (Rb) tumor suppressor activity, redirecting cellular processes toward differentiation and away from tumorigenesis.

In summary, our findings unveil p21 modulation, particularly through Nutlin 3A co-treatment, as a promising avenue for overcoming resistance in thyroid cancers, offering insights into novel therapeutic strategies to improve patient outcomes.

ANXA7, also known as synexin, has been shown to have a tumor suppressive role in glioblastoma, glioblastoma multiforme (GBM), melanoma, liver cancer, lung cancer, gastric cancer, nasopharyngeal carcinoma, colorectal cancer, ovarian cancer, breast cancer, prostate cancer and others ^16,17,19. Located on the human chromosome 10q21, ANXA7 is a member of the annexin superfamily⁴¹. This family is characterized by Ca ⁽²⁺⁾-regulation and phospholipid-binding proteins. Accumulated evidence indicates that the deregulation, loss of heterozygosity (LOH), and subcellular localization of ANXA7 are associated with the occurrence, invasion, metastasis, and progression of a variety of cancers ^33,36,37.

The BRAF gene is also referred to as proto-oncogene and the protein is more formally known as serine/threonine-protein kinase ^9,42. This protein plays a role in regulating the MAP kinase/ERK’s signaling pathway, which affects cell division and differentiation³². Diseases associated with BRAF mutation include melanoma, Cardio-faciocutaneous Syndrome, Lung Cancer and many other malignancies ^32,43,44. Evidence is also suggested that mutation of this protein is associated with over 60% of thyroid cancer⁷. B-type Raf (BRAF) kinase (V600E) mutation and p53 protein expression were evaluated in papillary thyroid cancer patients⁴⁵. Thyroid cancer is the most common endocrine malignancy, and most patients with thyroid cancer have a great chance for successful with treatment. There is, however, a group of patients with poor prognoses. The current research of thyroid tumor markers has related to identifying better diagnostic and prognostic circulating markers analysis (thyroglobulin, thyroid peroxidase, calcitonin, and carcinoembryonic antigen), cellular markers determination, and interpretation of results, as well. A number of molecular markers have been studied. Diagnostic value of some of them, e.g. TSHR, RET, RAS, is well known, while others have been investigated continually. Over-expression of BRAF and Met has been correlated with aggressiveness of the cancer. Markers said to be of prognostic value in thyroid cancer are CD82, c- myc and Plk-1. The combination of markers: galectin-3, fibronectin and HBME-1 have proven to be sensitive for differentiated thyroid cancer. The current data presents the well-known cellular marker of normal thyroid and parathyroid gland and correlation with thyroid cancer. Further studies on new cellular thyroid markers are essential for understanding and improving diagnostic and prognostic capabilities in thyroid cancer.

Based on our clinical findings, we decided to look at the drug sensitivity patterns in cell lines with similarly known mutations. The emergence of drug resistance and potential addiction in cancer treatment, particularly associated with MAPK/ERK pathway inhibitors like MEK or BRAF inhibitors, presents a significant challenge in oncology. The complexity is further compounded by the presence of multiple cancer-related mutations such as KRAS or p53 alongside the BRAF (V600E) mutation^9,28,46,47.

Developing strategies to overcome resistance and enhance sensitivity to these inhibitors is indeed crucial. One approach involves combination therapies targeting multiple pathways or mutations simultaneously. For instance, a combination of MEK or BRAF inhibitors with drugs targeting KRAS or p53 pathways might help address resistance mechanisms and improve treatment outcomes. Our focus was to identify novel connections for the improvement of BRAF gene mutation-based therapy. Our results showed that ANXA7, a known tumor suppressor gene that has been extensively studied by our lab for the past decade, is correlated to favorable outcomes. In this study we observed that the upregulation of ANXA7 causes drug induced chemo-sensitization to otherwise recalcitrant tumors with the BRAF V600E mutation. However, the exact mechanism which causes the tumor sensitization by ANXA7 expression remains to be clarified. The p21 induction has the same effect on the cell as the activation of p53, as it curtails the PI3-Akt mediated activation of NF-kB, which is a well characterized oncogenic driver in various cancers ⁴⁸. Previous study indicated that the bortezomib sensitivity increases by the combination of Nutlin 3 for the cancer cells having defective p53 expression⁴⁹. We have shown that ANXA7 appears to be a rapamycin-like endogenous regulator that can balance cell growth and survival in homeostasis and tumorigenesis through mTOR/Akt/PI3K pathway components ²⁶. This pathway along with mTOR axis is usually coactivated in BRAF mutations, which induces the vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR); these receptors are crucial in sustaining the tumor growth ⁵⁰. Overall, the ANXA7-mediated response is useful in the induction of pro-survival phenotypes shedding light on potential therapeutic strategies for tumors associated with BRAF mutations.

In our subsequent analysis, we tested the correlation of p21 to the ANXA7 response. This might point to the conclusion that the ANXA7 response as a tumor suppressor might be acting through a different mechanism other than cell cycle arrest. Interestingly, presence of the BRAF V600E mutation was directly correlated with ANXA7 expression. Thus, even though ANXA7 might not be directly related p21, it might be acting through BRAF-mediated downregulation of the ERK pathway, which results in c-MYC normalization. It is well-characterized that c-MYC is a driver of cancer in its mutated forms ⁵¹. Thus, drug therapy targeting p21 and ANXA7 upregulation will be a great tool in causing cell cycle arrest and increasing sensitivity of cancer cells to pharmacological agents. Our findings in this study suggest that the BRAF V600E mutation may be making the cells more invasive and more resistant by inhibiting the signaling of ANXA7. It is already known that the p53-mediated downregulation of p21 occurs in c-MYC overactivation ⁵². ANXA7 upregulation may thus chemo-sensitize the tumor if p21 modulators are added to the treatment regimen, as they will counteract the c-MYC overexpression and oncogenic drive towards stemness, which has the same effect as c-MYC inhibition in these conditions. This finding may be generalizable to a large degree of cancers including melanoma and thyroid cancers where BRAF V600E is highly prevalent (Fig. 1s and Fig. 2s). The relatively simple characterization of BRAF V600E mutation by PCR, and the expression levels by RT-PCR and western make these potential therapeutic strategies feasible for clinical translation.

To conclude, we believe that ANXA7 is an overlooked tumor suppressor which has an independent effect upstream of c-MYC. The implications of this are numerous: I) any modality which can upregulate ANXA7 could downregulate the mutated c-MYC oncogenic drive; II) upregulation of ANXA7 would automatically upregulate p21-mediated cell cycle arrest and tumor differentiation, and thus reduce the tumors to make them susceptible to chemotherapy; III) ANXA7 upregulation might have a direct role in chemo-sensitizing the tumor and overcoming drug non-responsiveness through the regulation of RB/p21 pathway.

The novelty in our current study lies in several key aspects related to cancer treatment related to BRAF V600E mutation and particularly thyroid cancer and melanoma. Specifically, our study unveils a previously unidentified pathway implicated in thyroid malignancy and drug resistance, involving the ANXA7/p21/BRAF/p53/MAPK axis. This novel pathway sheds light on the intricate interplay between ANXA7 expression, BRAF/p53 mutation, and their impact on disease progression and aggressiveness opening avenues for targeted therapeutic strategies.

Furthermore, our current research unveils insight into overcoming drug resistance. By investigating the interplay between ANXA7 expression and BRAF mutation, our study offers promising insights into overcoming resistance to BRAF or MAPK-targeting drugs in the treatment of thyroid cancer. We demonstrate that treatment with BRAF and MEK inhibitors leads to upregulated ANXA7 expression, decreased Ph-ERK levels, and increased apoptotic markers, indicating a potential strategy for overcoming drug resistance.

In addition, in this current study we discover a strategy for synergistic drug combinations to overcome the drug resistance. Our findings highlight the synergistic effects of combining drugs to elevate both p21 (Nutlin 3a) and ANXA7 levels, which enhances apoptotic signaling. Particularly, we observe a significant increase in bortezomib activity when combined with the p21 inducer Nutlin 3a molecule. This suggests a promising approach for enhancing the efficacy of existing therapies through targeted drug combinations.

Overall, our study offers novel insights into the molecular mechanisms underlying thyroid cancer progression and drug resistance, paving the way for the development of more effective therapeutic strategies for patients with BRAF-mutated thyroid cancer and potentially other malignancies.

Cells. Four thyroid cancer cell lines were chosen for the experiment - MDA-T68 acting as a wild type (lacking BRAF V600E), K1 and 8505C having the BRAF V600E mutation, and FTC-133 cells having the PTEN deletion mutation. The cells were grown in DMEM/F12 with 10% serum (FBS) or medium suggested by ATCC with the addition of penicillin and streptomycin to protect the culture from common contaminants. Cultures were periodically tested for mycoplasma contamination, and sterility was ensured. The incubation was done at 37^oC with 5% CO2 in a humidity-maintained environment.

Capillary Electrophoresis and Immunoassay: Capillary electrophoresis and immunoassay or Simple Western analyses were performed using the Wes machine (ProteinSimple, Bio-Techne, San Jose, CA) according to the manufacturer’s protocol ^53,54 ⁵⁵. Briefly, 1 µg total lysate samples were mixed with a master mix (ProteinSimple) to a final concentration of 1x sample buffer, 1x fluorescent molecular weight markers, and 40 mM dithiothreitol (DTT), and the samples were then heated at 80 C for 5 min. The samples, blocking reagent, primary antibodies, HRP-conjugated secondary antibodies, chemiluminescent substrate, and separation and stacking matrices were dispensed to designated wells in a pre-designed plate (ProteinSimple). After plate loading, the separation electrophoresis and immune-detection steps took place in the capillary system and were fully automated. Simple Western analysis was carried out at room temperature and instrument default settings with high dynamic range (HDR) for the chemiluminencent detection step. The details of antibodies and their corresponding dilutions were identified and listed in Table 1. The digital images were analyzed with Compass software (Protein Simple), and the quantified data of the detected protein were reported in molecular weight.

Database, Bioinformatics analysis, and Statistics: We performed mutational analysis using cBioportal [http://www.cbioportal.org] ^56,57. The results were reported in color-coded figures, which were based on an odds ratio of the presence of mutational frequency to the specific cancer type. The Cancer Cell Line Encyclopedia (CCLE - https://portals.broadinstitute.org/ccle) ⁵⁸ database was used to study the transcriptomic relationship between BRAF and ANXA7. Relative protein abundance was calculated for BRAF and ANXA7 using the MD Anderson cell line project (MCLP, https://bioinformatics.mdanderson.org/main/MCLP:Overview) ³¹.

Drugs/inhibitors. Most of the inhibitors were purchased from www.selleckchem.com and Nutlin-3A was purchased from Sigma-Aldrich. The drug concentrations and other details were as described in Table 2.

Annexin A7 (ANXA7), cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 (p21), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), Mitogen-Activated Protein Kinase (MAPK), Extracellular signal-Regulated Kinase (ERK), Phosphorylated-ERK (Ph-ERK), Phosphoinositide 3-kinase (PI3K), Protein Kinase B (Akt), Rat sarcoma viral oncogene homolog (Ras), Rapidly Accelerated Fibrosarcoma (Raf), Mitogen-Activated Protein Kinase Kinase (MEK), Extracellular signal-Regulated Kinase (Erk), and Signal Transducer and Activator of Transcription (STAT).

Statement of Data Availability:

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Acknowledgements:

This work was supported by the Collaborative Health Initiative Research Program (CHIRP) funding (CHIRP ID# IAA-A-HL-14-007). The opinions or assertions contained herein are those of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.

Author Contributions

Conceptualization: A.B., H.P., M.S.

Data collection and curation: A.B., M.Su., N.P., S. R., E. R., N. G., M.S.

Formal analysis: A.B., M.S., N.P., S.R., E.R.

Funding acquisition: H.P., M.S.

Investigation: A.B., M.S.

Methodology: A.B., M.S.

Project administration: A.B., M.S.

Resources: A.B., M.S.

Software: A.B., N.P., E.R.

Supervision: A.B., H.P., M.S.

Validation: A.B., N.P., S.R., E.R.

Visualization: A.B., M.S.

Writing – original draft: A.B., M.Su, M.S..

Writing – review & editing: A.B., M.Su, N.P., S. R., E. R., N. G., H.P., M.S.

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Role of Annexin 7 (ANXA7) as a tumor suppressor and a regulator of drug resistance in thyroid cancer

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