Elevated of HSP90 associates with expression of EGFR levels and predicts poor prognosis in non-small cell lung cancer patients

doi:10.21203/rs.3.rs-2101621/v1

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Elevated of HSP90 associates with expression of EGFR levels and predicts poor prognosis in non-small cell lung cancer patients

https://doi.org/10.21203/rs.3.rs-2101621/v1

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Background

The molecular chaperone protein HSP90 is a crucial mediator of proteostasis in eukaryotic cells under various stress conditions. EGFR participates in signal transduction relating to tumorigenesis and progression. The relationship between the combined expression of HSP90 and EGFR in non-small cell lung cancer (NSCLC) and clinicopathological features and long-term prognosis is not clear.

Methods

We analyzed The Cancer Genome Atlas (TCGA) gene data from Xiantao platform and GEPIA2 to study the expression of HSP90 and EGFR on mRNA level and their correlation in patients with NSCLC. Besides, 53 normal lung samples and 352 paraffin-embedded NSCLC were selected randomly for immunohistochemical (IHC) staining with anti-HSP90 and anti-EGFR antibodies, respectively.

Results

The TCGA data revealed that HSP90 and EGFR expression on mRNA and protein was significantly higher in NSCLC patients than non-cancerous lung tissues (Non-CLT) (both p < 0.05). Moreover, increased HSP90 showed a positively correlation with EGFR in mRNA levels (both p < 0.0001). Higher HSP90 expression appeared in lymph node metastasis (LNM) or advanced clinical stage of NSCLC (p = 0.019, p = 0.013, respectively). The survival rate of high expression of HSP90 and EGFR alone or in combination was lower than that of other phenotypes in NSCLC (p < 0.001, P = 0.011, p < 0.001, respectively). In multivariate Cox proportional hazard regression analysis, HSP90 and EGFR were further identified as independent poor prognostic factors in NSCLC.

Conclusions

Elevated HSP90 and EGFR might suggest a synergistical role in promoting the development in patients with NSCLC. High expression of these two proteins might be novel prognostic biomarker for NSCLC, who provides potential application value for NSCLC targeted therapy.

HSP90

EGFR

non-small cell lung cancer

targeted therapy

prognosis

Lung cancer is one of the most commonly diagnosed malignant tumor which remains the leading cause of cancer deaths in the world to date [1]. NSCLC includes lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC) in histological types and accounts for 85 percent of all lung cancers [2]. Early lung cancer can be treated by surgical resection; unfortunately, most lung cancer patients have a less than 20% 5-year survival rate for their first diagnosis already at its advanced stage [3]. Lung cancer is universally recognized as a complicated and heterogeneous disease with various signaling pathways [4]. Hence, far-reaching research on molecular biomarkers for NSCLC is critical in identifying the disease's malignant, assessing prognosis, and looking for new therapeutic targets, all of which help to guide more effective treatment and lower mortality.

Heat shock proteins (HSPs) are a group of highly conserved proteins that maintain cellular homeostasis to deal with various complicated stresses, for instance, high temperature, hypoxia or chemical agents [5]. As an important member of the heat shock family, heat shock protein 90 (HSP90) was further established to have important roles within various biological procedures, mainly protein synthesis, cell apoptosis and cytoskeleton maintenance [6]. The human HSP90 family consists of four members, including two main isoforms HSP90α and HSP90β, GRP94 and TNF receptor-associated protein 1 (TRAP1), respectively encoded by HSP90AA1, HSP90AB1, HSP90B1, and TRAP1 [7]. Previous work has shown that overexpression of HSP90 exists in a variety of malignancies like nasopharyngeal, hepatocellular, colorectal carcinoma and so on, which is related to the poor prognosis for patients [8–10] and leads to rapidly development of HSP90 inhibitors for targeted therapy.

Epidermal growth factor receptor (EGFR), which is a type of tyrosine kinase, involved in the signal transduction relating to tumorigenesis and progression [11]. Great progress has been made in the precise treatment of EGFR gene, however, not all patients with NSCLC are sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Furthermore, even those who respond to EGFR-TKIs eventually develop resistance to it [12]. Consequently, the development of resistance to these drugs has become a major clinical problem over the years. Thus, identification of new biomarkers and development of new inhibitors combine with EGFR-TKIs might be new therapeutic strategy to find more effective treatment of NSCLC patients.

Given what we know, several reports have linked individual expression of HSP90 and EGFR proteins to clinicopathological features of NSCLC patients. To date, nevertheless, the specific association between HSP90 and EGFR in NSCLC remains unknown. We investigated the IHC expression of HSP90 and EGFR in 53 normal lung cases and 352 NSCLC samples, and further analyzed the correlation between HSP90 and EGFR expression, clinicopathological characteristics and prognostic significance.

2.1 TCGA database

We used the Xiantao Xueshu web tool (https://www.xiantao.love/products) to obtain 108 NC tissues and 1037 NSCLC tissues from the TCGA database for tumor/normal differential expression of HSP90 and EGFR and performed their correlation analysis in GEPIA2 (http://gepia2.cancer-pku.cn/#index).

2.2 Ethical statement

The Ethics Review Board of Central South University's Second Xiangya Hospital authorized all protocols. (Scientific and Research Ethics Committee, No. S039/2011). The individuals gave their informed consent before surgery, and every research sample was taken written consent as well. For the minors, however, the guardian must sign a written consent on their behalf.

2.3 Clinical data and tissue microarrays (TMA)

We acquired 53 Non-CLT cases and 352 paraffin-embedded NSCLC, including SCC (159) and ADC (193), from the Department of pathology of Xiangya Second Hospital of Central South University. These tissues were resected by the Thoracic Surgery Department of the Second Xiangya Hospital between 2003 to 2013. All patients’ diagnoses were histologically confirmed by experienced pathologists and NSCLC patients who received chemotherapy or radiotherapy before surgery were excluded. We took the time begin with the initial diagnosed day to the last follow-up as the definition of overall survival time. Out of the 352 patients considered, 204 (58.0%) stage III cases, 72 (20.4%) stage II cases, and 76 (21.6%) stage I cases were confirmed. The final follow-up results showed that 191 patients survived and 161 patients died. The pathological diagnosis were performed in accordance with the WHO histological classification and the clinical staging of cases were based on the Eighth Edition Lung Cancer Stage Classification [13]. Detailed clinicopathological data (Supplement Table 1) were retrieved from relevant pathology records. Tissue microarrays were fabricated with two tumor cores in TMAs and two normal lung cores included in Non-CLT TMAs by applying the TMA technology according to the formerly method [14]. The average perforation diameter was 0.6 mm.

2.4 Antibody selections

The staining condition of primary antibody as follows: HSP90 (1:1000, rabbit polyclonal antibody, Catalog: #4877; Cell Signaling Technology, Danvers, MA, USA) and EGFR (1:200, Novocastra NCL-EGFR-384 clone, Leica Biosystems Newcastle, Newcastle Upon Tyne, UK) followed by overnight at 4°C.

2.5 Immunohistochemistry (IHC) and scores

The IHC staining for HSP90 and EGFR in TMAs of NSCLC and non-CLT patients was performed according to the previous method [15]. Each experiment was added positive and negative control slide to validate the IHC staining and the IgG isotype-matched antibody was the negative contrast which can help decide the specificity of the antibody in IHC.

Two pathologists (SF and QW) independently evaluated all staining of TMA sections at 200× magnification light microscopy. The total expression marks according to the distribution and intensity for each IHC staining was determined as following formula: overall score = intensity mark + percentage mark. Briefly, the intensity of staining for HSP90 and EGFR was visually graded as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Meanwhile, the percentage scores of positive tumor cells were assigned as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%). Thus, the total score of HSP90 and EGFR proteins levels in all samples was between 0 and 7. In addition, according to the log-rank test heterogeneity measurement of overall survival (OS) in patients with NSCLC, the optimal critical levels of the two proteins were 5 and 2, respectively. For the final analysis, total scores could be separated into two parts: a score above 5 and 2 was regarded as positive/high expression of the two proteins, and the others were categorized low expression. All IHC scoring discordances were resolved through consensus by two pathologists and the agreement between them is 95%.

2.6 Statistical analysis

We used SPSS 24.0 software to conduct statistical analysis. Chi square test was applied to explore the relationship between HSP90 and EGFR expression and clinicopathological features in NSCLC, and the association between HSP90 and EGFR and the clinicopathological characteristic in NSCLC. Furthermore, the study used Spearman’s rank correlation coefficient, Kaplan-Meier analysis and log-rank test to assess the correlation between HSP90 and EGFR, survival rate and analysis of survival rate curve, respectively. Analysis was performed with the Cox proportional hazards regression model to assess independent prognostic factors for survival. All p values were two-sided, and p < 0.05 was utilized as a threshold value for statistical significance.

3.1 Bioinformatics analysis of HSP90 and EGFR in NSCLC

Based on the TCGA database, we selected numbered 108 NC tissues and 1037 NSCLC tissues. The results demonstrated that EGFR and two main isoforms of HSP90 mRNA expression were significantly higher in NSCLC tissues compared with in NC. (both P < 0.05). Consistent with this, HSP90 was also positively correlated with EGFR mRNA level in patient tumors (r = 0.23, r = 0.28, both P < 0.0001; Figure 1)

3.2 Expression of HSP90 and EGFR increased significantly in NSCLC

In this study, we performed IHC to detect the subcellular localization and positive expression of HSP90 and EGFR in resected NSCLC and Non-CLT tissues. We found positive staining of HSP90 and EGFR in the cytoplasm of cancer cells (Figure. 2 A and B, D and E), but were not found in Non-CLT (Figure 2 C, F). The percentages of high expression of HSP90 and EGFR proteins was 56.6% (90/159) and 35.2% (56/159) in lung SCC, 67.9% (131/193) and 58.0% (112/193) in lung ADC, respectively. And the data in Non-CLT was 11.3% (6/53) and 26.4% (14/53). The results were illustrated in Figure 3 that the level of HSP90 and EGFR expression in NSCLC was notably up-grading (all P < 0.001).

3.3 Association between expression of proteins and clinicopathological features of NSCLC

Furthermore, the study also used chi-square test to investigate the relationship with regard to age, gender, clinical stage, lymph node metastasis (LNM) status, histological type, pathological degree, and survival status and elevated expression of HSP90, EGFR and the combination of these two proteins. As showed in Table 1, overexpression of HSP90 protein was positively correlated with the clinical stage (P = 0.019) and no marked correlation was found between EGFR or the combination of HSP90 and EGFR expression and clinical stage (P > 0.05). Tissues with LNM status in NSCLC had higher expression of HSP90 and the combination of these two proteins than that without LNM (P = 0.013, P = 0.009, respectively). The individual or combined expression of HSP90 and EGFR in lung ADC tissues was evidently increased than that in lung SCC cases (all P < 0.05). Besides, the data showed that high expression of HSP90 and EGFR alone or in combination had a lower incidence of OS than other cellular phenotypes of HSP90 and EGFR in NSCLC (all P < 0.05). However, no prognostic significance was discovered with age, gender and pathological degree in patients with NSCLC (all P > 0.05).

3.4 Correlation between HSP90 and EGFR expression in NSCLC

We used these data to explore the correlation between HSP90 and EGFR expression in NSCLC. It is apparent from Table 2 that elevated HSP90 expression level was positively correlated with EGFR expression level in patients with NSCLC (r = 0.183, P < 0.001, respectively).

3.5 Status of expression of HSP90 and EGFR on patients’ prognosis

Kaplan-Meier survival curves revealed the relationship between HSP90, EGFR, combined expression of HSP90 and EGFR, OS rate and clinicopathological features in univariate survival analysis. And we also analyzed the statistical significance using the log-rank test. Up-regulated HSP90 patients had lower overall survival rates than those with down-regulated HSP90 (P < 0.001) (Figure 4A & Table 3), as was found with increased EGFR (P = 0.011) (Figure 4B & Table 3). Meanwhile, patients with raised combined high expression of HSP90 and EGFR (Figure 4C, P < 0.001) also have lowest survival rates than others. In addition, clinical stages I and II (P < 0.001) (Figure 4D & Table 3) or without LNM (P < 0.001) (Figure 4E & Table 3) had longer survival time than those with stages III or LNM. Moreover, higher OS rates could be found within these well and moderated differentiation NSCLC patients, rather than the poor differentiation patients (P = 0.003) (Figure 4F & Table 3).

From Cox proportional hazard regression, the results in Table 3 exhibited that overexpression of HSP90 might significantly serve as an independent poor prognostic factor for patients with NSCLC (P = 0.009), adding to EGFR (P = 0.034), clinical stage (P = 0.001) and pathological degree (P = 0.007). Nevertheless, we could confirm that age, gender, LNM status and histological type were no prognostic significance in NSCLC patients (Table 3, all P > 0.05).

In our current research, we observed that HSP90 expression in NSCLC patients was elevated significantly both in mRNA and protein levels compared to NC tissues. Higher HSP90 expression appeared in patients with advanced clinical stage and LNM. Moreover, NSCLC patients with up-expressed HSP90 had a lower survival rate than that with down-expressed HSP90. Taking together, these data suggested that overexpression of HSP90 might take an active part in the occurrence and development of NSCLC. Several research proved that aberrant expression of HSP90 is related to the development and progression of multiple human malignant tumors involves breast cancer, lung cancer and pancreatic cancer, and relating to the poor prognosis of patients, which confirmed its vital role in various cancers [16-18]. As one of the most abundant molecular chaperones, HSP90 can protect cancer cells under adverse physiological and stress conditions such as hypoxia, nutritional deficiency, chemicals and low pH, to protect mutated substrate oncoproteins from misfolding and degradation and allows their malignant transformation [19]. Our data revealed that the survival time of patients high HSP90 is shorter than that with low expression and HSP90 could be used as an independent prognostic indicator of NSCLC.

The data pointed to a positive correlation between HSP90 and EGFR in both mRNA and protein levels. Cases of Lung ADC had evidently increased individual or combination expression of HSP90 and EGFR than in Lung SCC. High co-expression of HSP90 and EGFR of NSCLC patients also experienced poorer clinical outcomes than other phenotypes of these two proteins, as shown by the lower OS rates. Furthermore, the observation of high HSP90 and EGFR expression suggested poor independent prognostic factors for NSCLC patients survival. The data suggested that there might be a crosslink between HSP90 and EGFR signal pathways. It is reported that EGFR is a typical oncogenic client protein of HSP90 which may be involved in regulating the ATP ase‑associated conformational changes of the HSP90 dimer that occur during the procession, and maintaining the stability of oncoproteins [20]. Of note, the association of HSP90 with EGFR could be mediated through the N-terminal ATPase domain of HSP90, and the activity would further be modulated via binding with co-chaperones, which could explain the positive correlation between HSP90 and EGFR expression [21]. Moreover, co-expression of HSP90 and EGFR might produce a congenerous effect on maintaining the stability of oncoproteins and further participated in accelerating growth, proliferation and survival of NSCLC cells [22]. On one hand, owing to EGFR is a highly sensitive client protein of HSP90, it has been proved that proopiomelanocortin (POMC) transcription mediated by the MAPK pathway reduced and the function of EGFR could be constrained after inhibition of HSP90 expression [23, 24]. On the other hand, the binding of FM807 to the N-terminal of HSP90 can destroy the HSP90 / client complex and degrade EGFR, thereby inhibiting the downstream Raf / MEK / ERK and PI3K / Akt pathways which promoting that HSP90 and EGFR work [25]. As mentioned above, the regulatory interaction network of HSP90 is still of great intricacy, and further studies may be needed to reveal the potential mechanisation of the relationship between HSP90 and EGFR later on [26].

It has been well established that EGFR is one of the first reasonably selected molecules for targeted therapy in patients with NSCLC and promotes the development and progression of NSCLC [27]. EGFR-TKI-based molecular targeted therapy has shown clinical efficacy in the treatment of resected NSCLC [28]. The first-generation EGFR-TKI (gefitinib), the second-generation EGFR-TKI (dacomitinib) and third-generation EGFR-TKI (osimertinib) possessed significant effect on NSCLC patients with EGFR mutation, have been commonly used as a major anticancer in the treatment of NSCLC [29, 30]. To deal with the drug resistance of the first and second generation EGFR-TKI, the third-generation EGFR-TKIs has been approved to deal with the resistance associated with the EGFR T790M mutation, which is tipped to provide significant survival benefits for advanced NSCLC [31]. Regardless of the significant progress of EGFR inhibitor treatment, constitutive and resistance to EGFR inhibitors remains a major challenge in cancer treatment [32].

In view of the close relationship between HSP90 and the molecular coordination of cancer occurrence and progression, the development of HSP90 inhibitors provide a promising research direction for targeted cancer therapy [33]. Geldanamycin derivatives, resorcinol derivatives, and other synthetic HSP90 inhibitors have been discovered and have entered into clinical evaluation since 2000 [34]. Although it has a strong antitumor effect, the current results of HSP90 inhibitors in the clinical stage are not satisfactory due to intolerable adverse reactions such as cardiotoxicity, ocular toxicity and hepatotoxicity, as well as drug resistance [35]. Taken together, considering the potential resistance to EGFR-TKI and toxic side effects of HSP90 inhibitors for NSCLC patients, combination of drugs seems particularly promising. It is reported that preclinical and early-phase clinical studies have demonstrated the antitumor activity of HSP90 inhibitors when used in combination with chemotherapy or targeted agents, despite the clinical efficacy of monotherapy is limited [36]. Surprisingly, targeted co-inhibition of both HSP90 and EGFR is considered particularly promising therapies for better efficacy in the treatment of NSCLC patients and HSP90 inhibition could overcome EGFR amplification-induced resistance to third-generation EGFR-TKIs [37], which suggests that combined inhibition of HSP90 and EGFR could be a better regimen than single targeted inhibition.

Acknowledgements

Not applicable.

Author contribution statement

Conceived and designed the experiments: Qiuyuan Wen. Performed the experiments: Ying Zhou and Yaoxiang Tang. Analyzed the data: Jiadi Luo, Yang Yang and Hongjing Zang. Prepared figures and tables: Yue Ning and Hongmei Zheng. Wrote the manuscript: Ying Zhou. Revised the manuscript: Songqing Fan and Qiuyuan Wen.

Funding

The work was supported by the Natural Science Foundation of Hunan Province (grant No. 2017JJ3457), Hunan Provincial Innovation Foundation for Postgraduate (grant No. CX20210373), the Fundamental Research Funds for the Central Universities of Central South University (grant No. 2021zzts1045) and the National Natural Science Foundation of China (grant No. 81703009, 81972838 and 82102805).

Data availability statement

Raw dates will be found on: https://doi.org/10.6084/m9.figshare.19918294.v2

Ethics approval and consent to participate

In this study, all procedures performed on research samples were approved by the ethical standards of the Institute Ethics Committee of the Second Xiangya Hospital of Central South University (approval No. S039/2011). All methods were performed in accordance with the 1964 Helsinki declaration and its later amendments of comparable ethical standards. Written Informed consent was obtained from all subjects or their legal guardian.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.

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Table 1. Analysis of the association between expression of HSP90 and EGFR and clinicopathological features of NSCLC (n=352)

Clinicopathological features (n)	HSP90			EGFR			HSP90/ EGFR^#
Clinicopathological features (n)	High (%)	Low (%)	P-value	High (%)	Low (%)	P-value	P⁺ (%)	N^- (%)	P-value
Age(years)
≤50 (n=100)	68(68.0)	32(32.0)		48(48.0)	52(52.0)		40(40.0)	60(60.0)
＞50 (n=252)	153(60.7)	99(39.3)	0.223	120(47.6)	132(52.4)	1.000	81(32.1)	171(67.9)	0.172
Gender
Male(n=267)	167(52.5)	100(37.5)		124(46.4)	143(53.6)		93(34.8)	174(65.2)
Female(n=85)	54(63.5)	31(36.5)	0.898	44(51.8)	41(48.2)	0.455	28(32.9)	57(67.1)	0.794
Clinical stages
Stage _Ⅰ-Ⅱ (n=148)	82(55.4)	66(44.6)		69(46.6)	79(53.4)		43(29.1)	105(70.9)
Stage _Ⅲ(n=204)	139(68.1)	65(31.9)	0.019*	99(48.5)	105(51.5)	0.747	78(38.2)	126(61.8)	0.088
LN status
LNM (n=211)	144(68.2)	67(31.8)		105(49.8)	106(50.2)		84(39.8)	127(60.2)
No LNM (n=141)	77(54.6)	64(45.4)	0.013*	63(44.7)	78(55.3)	0.384	37(26.2)	104(73.8)	0.009*
Histological type
SCC(N=159)	90(56.6)	69(43.4)		56(35.2)	103(64.8)		34(21.4)	125(78.6)
ADC(N=193)	131(67.9)	62(32.1)	0.035*	112(58.0)	81(42.0)	0.000*	87(45.1)	106(54.9)	0.000*
Pathological grade
Well/Moderate(n=151)	90(59.6)	61(40.4)		76(50.3)	75(49.7)		54(35.8)	97(64.2)
Poor (n=201)	131(65.2)	70(34.8)	0.316	92(45.8)	109(54.2)	0.451	67(3433)	134(66.7)	0.652
Survival status
Alive(n=191)	104(54.5)	87(45.5)		76(39.8)	115(60.2)		45(23.6)	146(76.4)
Dead(n=161)	117(72.7)	44(27.3)	0.001*	92(57.1)	69(42.9)	0.001*	76(47.2)	85(52.8)	0.000*

*Chi-square test, statistically significant difference (P< 0.05). Abbreviations: LNM, lymph node metastasis; SCC, squamous cell carcinoma; HSP90/EGFR^#P⁺, common high expression of HSP90 and EGFR, P^-, other combination of expression of these two proteins.

Table 2. The pairwise association between expression of HSP90 and EGFR in NSCLC

	HSP90
EGFR	0.183 0.001*
r
Sig. (2-tailed)

*Spearman’s rank correlation test, statistically significant difference. * P < 0.05.

Table 3. Summary of univariate/multivariate analysis for overall survival in patients with NSCLC (n=352)

Variables	Univariate analysis			Multivariate analysis
Variables	Average survival time (SE)	95%CI	P-value	Exp (B)	95.0%CI	P-value
HSP90
High expression	45.003(2.753)	39.608-50.398	0.000*	1.615	1.129-2.310	0.009*
Low expression	78.069(5.470)	67.349-88.790
EGFR
High expression	48.174(3.451)	41.410-54.938	0.011*	1.425	1.027-1.976	0.034*
Low expression	70.063(4.953)	60.355-79.772
Clinical stages
Stage _Ⅰ-Ⅱ	68.935(4.220)	60.664-77.207	0.000*	1.898	1.284-2.806	0.001*
Stage _Ⅲ	49.168(3.862)	41.597-56.738
LN status
LNM	46.858(3.248)	40.491-53.225	0.000*	1.414	0.964-2.074	0.076
No LNM	73.673(5.884)	62.140-85.207
Histological type
SCC	63.040(5.770)	51.730-74.350	0.407	1.050	0.745-1.480	0.779
ADC	52.076(3.415)	45.383-58.768
Pathological grade
Well/moderated	59.352(3.539)	52.415-66.289	0.003*	1.503	1.077-2.099	0.007*
Poor	55.144(4.150)	47.010-63.278
Age
≤50	46.513(3.641)	39.377-53.650	0.448	1.012	0.718-1.427	0.945
＞50	61.801(4.091)	53.783-69.819
Gender
Female	53.896(4.450)	45.174-62.619	0.366	0.875	0.590-1.297	0.505
Male	58.731(3.929)	51.031-66.431

Abbreviations: CI, confidence interval; Exp(B), odds ratio; SE, standard error; LNM, lymph node metastasis;

* P< 0.05.

No competing interests reported.

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Elevated of HSP90 associates with expression of EGFR levels and predicts poor prognosis in non-small cell lung cancer patients

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