TIMPs are 21 kDa proteins of 184–194 amino acids, and the human genome has four paralogous genes that encode for TIMP 1–4. TIMPs can inhibit MMPs, ADAMS, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). TIMPs are undoubtedly crucial for regulating the metalloproteinases which can break down the extracellular matrix and exfoliated cell surface components[27]. TIMP abnormalities have been consistently found in human malignancies in the comprehensive quest for genetic markers of tumor progression.
TIMP is generally localized in the extracellular matrix, which plays a crucial role in extracellular matrix degradation, angiogenesis, and immune response[28]. Recent studies have confirmed that TIMP has a tumor-promoting effect, and the high expression of TIMP is associated with a poor prognosis of almost all cancers, although TIMP was traditionally considered as a tumor suppressor[29]. The diversity of TIMP's functions suggests the complexity of its origins, therefore, it is imperative to conduct a thorough investigation into the source of TIMP for targeted therapy. Currently, several studies have demonstrated that TIMP in tumor tissues comes from tumor cells[30]. In highly lethal PDAC, the interaction of TIMP1, from tumor cells, with its receptor CD63 could trigger Neutrophil Extracellular Trap Formation in Pancreatic Cancer through the ERK signaling[31]. Exosome miR-3157-3p, released by cancer cells creating tumor microenvironment, could inhibit the expression of TIMP, and regulate the expression of VEGF/MMP2/MMP9 and occludin in endothelial cells, hence promoting angiogenesis in NSCLC[32]. Our PCR results indicated the potential source of tumor cells, nevertheless, it is noteworthy that the abnormal expression of TIMP in matrix cells within the extracellular matrix is also associated with tumor progression, in addition to the tumor cells themselves. The recent research illustrated that high expression of TIMP1 in tumor-related fibroblasts in lung adenocarcinoma tissues could encourage tumor cell invasion, whereas co-culturing fibroblasts with tumor tissues carrying low expression of TIMP might discourage the invasion[29]. It is obvious that TIMP is more than just a tumor promoter; understanding its source diversity and different function in tumor progression would enable us to explore the better prognostic and therapeutic targets.Therefore, to better understand the mechanisms underlying TIMP gene family expression in cancer, we focused on the expression features and prognostic value of the TIMP gene family in pan-cancer. According to the expression levels in TCGA, we revealed the expression of the TIMP gene family varied among different human tissues. In terms of tumor tissues, TIMP1 expression was relatively higher in most cancers than in normal tissues while TIMP3 expression was downregulated. For others, TIMP2 expression declined in 18 types of cancers and elevated in 9 types of malignancies. The result indicated that TIMP2 played a detrimental role in ACC, LUSC, and SKCM, whereas low expression of TIMP2 was relevant to a high survival probability in BLCA, CESC, UCEC, LUSC, TGCT, and STAD. At the same time, TIMP4 expression was considerably reduced in 21 different types of tumors, and it was accompanied by a poor prognosis (HR > 1, p < 0.05) in the ACC, HNSC, THCA, UCS, and UVM. We utilized the UALCAN database for the protein expression of the TIMP gene family in pan-cancer. However, the database only contains the data of TIMP1, TIMP2, and TIMP3 protein expression in GBM, PAAD, and UCEC. When comparing the protein expression with the gene expression, we discovered that the results were consistent. The gene and protein expressions of TIMP1, TIMP2 and TIMP3 were upregulated in GBM and PAAD. In UCEC, the gene and protein expressions of TIMP1 were increased, whereas TIMP2 and TIMP3 were decreased. The analysis of genes in clinicopathology also confirmed the prognostic value. Due to the high frequency of gene mutations in tumors, we analyzed the mutation profile of TIMP in cBioPortal. All in all, TIMP family gene alteration frequency was less than 5% in most malignancies and TIMP1 was the most.
We observed that the expressions of TIMP gene family vary depending on the tumor stage. With the development of BLCA, the expression of TIMP2 increased gradually, and the expression of TIMP4 were also upregulated with the advancement of TGCT. Therefore, we speculated that the expression of high TIMP4 is not only related to the early stage of TGCT, but also to the advanced stage. Additionally, when comparing the protein expression with the gene expression, we discovered that the results were consistent in GBM, PAAD, and UCEC, however, our study is subject to certain limitations, and extra clinical data from patients may be necessary to validate this conclusion.
DNA alterations play an essential role in tumor progression, and the presence of TIMP mutations exists in numerous tumors, indicating a crucial function for TIMP gene family mutations in tumor development[33]. TIMP is conservative in multiple tissues of human body, and TIMP mutations have a great impact on its function in pathological state[34]. According to our results, the mutation frequencies of TIMP1, TIMP2, and TIMP3 in diverse malignancies are below 5%, whereas the frequency of TIMP4 is the highest (12%). The mutation of TIMP2 could amplify a proteolytic cascade driven by interactions of progelatinase A (proMMP-2) and membrane-type matrix metalloproteinase 1 (MT1-MMP), thereby speeding up the degradation of extracellular matrix[35]. Moreover, the C-terminal domain of TIMP is of vital importance to its affinity with MMP, and any alterations in this sequence will result in a modification of the interaction between MMPs and TIMP[36, 37]. The mutation of TIMP is a key contributor in the pathogenesis of diseases, but its comprehensive roles in tumorigenesis remain to be explored[38]. Despite of this, gene mutation has tremendous prospects in the diagnosis, prognosis and treatment of malignancies, especially in hepatocellular carcinoma, invasive breast cancer, uterine carcinosarcoma, and urothelial bladder cancer[39–42]. These findings implied that directing interventions towards these modification targets is expected to decrease the dysfunction of TIMP, which could potentially serve as the underlying mechanism for instigating TIMP to facilitate tumorigenesis.
As is known, tumor immune microenvironments are meaningful to cancer therapy. TME is composed of various components, such as tumor stromal cells, immunocyte, acellular components and so on[43]. Tumor-associated macrophages (TAMs) are the most abundant tumor-infiltrating immune cell, as indispensable elements in the TME[43]. TAMs had phenotypic plasticity and pronounced heterogeneity, being able to develop a distinct phenotype in response to local microenvironmental stimulation[44]. There are two major directions of phenotypic and functional macrophage polarization, M1 and M2. M1 macrophages represent reliable phagocytosis, which may stimulate the immune response and play an anti-tumor role by releasing proinflammatory factors. However, M2 macrophages can generate immunosuppressive cytokines, accelerate the transformation from normal cells to tumor cell and promote the cancer development[45]. Further, TAMs are capable of inducing immune tolerance, a complex process, in which T lymphocytes and other cells also play considerable roles[46]. We explored the relationship between TIMPs and immune cells, and the result showed that the TIMP gene family played an essential role in immunity via the "CIBERSORT" algorithm. TIMP1 is positively correlated with the level of M2 macrophage infiltration in certain malignancies, and M2 macrophages can promote the progression of cancers. It has been reported that TIMP1 has a clear correlation with the poor prognosis of nearly all types of cancer. Therefore, we hypothesized that TIMP1 might facilitate the occurrence and development of tumors by modulating M2 macrophage infiltration levels[30]. Likewise, the expression of TIMP1 is associated with T cells, B cells, NK cells, and other immune cells, which are involved in innate and adaptive immune responses, as integral parts of carcinoma[47]. In certain types of cancer, there exists a positive correlation between TIMP2 and M2 phenotype, however, the expression level of TIMP2 and its prognostic trends have heterogeneity between different kinds of cancers[30]. TIMP3 is widely regarded as a tumor-suppressive protein, and the downregulation of TIMP3 is linked to poor prognosis[30, 48]. The studies were consistent with prior research indicating that TIMP3 could induce apoptosis and reduce invasion and metastasis in some cancers[30, 49, 50]. Similarly, we observed a positive correlation between TIMP3 and M2 infiltration in a few tumors, which may provide a reasonable explanation for the anti-cancer impact of TIMP3. TIMP expression was constant in unstimulated human peripheral blood monocytes, B cells, and T cells. TIMP1, TIMP2, and TIMP4 were much more abundantly expressed in monocytes than in B or T cells, whereas TIMP3 was overexpressed in B cells[51]. What’s more, TMB and MSI were considered as the common biomarker for immunotherapy, therefore the connection between TMB, MSI, and TIMPs also illustrated the impact of TIMPs in immune-checkpoint-inhibitors (ICIs). To figure out the potential regulatory mechanisms for TIMPs in pan-cancer, GO and KEGG analyses were utilized. GO analysis based on the TIMP gene family was enriched in metabolism, biological control, and developmental process. KEGG pathway enrichment analysis of the TIMP gene family was collected in bladder cancer, IL-17 signal pathway, parathyroid hormone synthesis, section and action, GnRH signal pathway, and so on.
Altogether, TIMPs firmly participated in the development and process of the pan-cancer hallmarks and may serve as promising biomarkers for cancer immune therapy. Our analysis results seem to be consistent with numerous other studies. Ma et al. found that TIMP1 stimulated cell proliferation and invasion through the TIMP1/FAK/Akt pathway in right-sided colon cancers, leading to a poor prognosis[52]. Guccini reported that TIMP1 deletion rewired the senescence-associated secretory phenotype (SASP) to encourage cell invasion and migration in animal models of prostate cancer[53]. Mendes et al. discovered that when TIMP2 was transfected into ENU1564, rat breast cancer cells, the animals exhibited a drop in situ growth in tumors, a decline in situ metastasis, and no metastasis in the brain, which was owing to the lower MMP2 activity[54]. TIMP3 was considerably downregulated in breast cancer, which was on account of the elevated expression of MSI1 in cancer tissues. MSI1 could block TIMP3, increasing the degree of MMP9 transcription to promote breast cancer metastasis[55]. The capacity of breast cancer cells to invade was inhibited by overexpressing TIMP4. It emphasized the therapeutic potential of TIMP4 in treating the malignant evolution of cancer[56]. Above all, the abnormal of TIMPs expression in targeted cancer probably played a crucial part in the occurrence of cancers. Due to the importance of immunotherapy, the relationship between TIMP2 and immunity has also been studied. Wang et al. demonstrated that TIMP2 is associated with the immune microenvironment[57]. In GBM, TIMPs were displayed to correlate with the infiltration of various immune cells, including CD4 + T cells, macrophages, neutrophils, B cells, CD8 + T cells, and dendritic cells[58]. TIMPs were lower expressed in innate immunity and metamorphosis[59]. More specifically, TIMPs had a potential role in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization[60]. Insect research also demonstrated the multiple roles of TIMPs in immunity and metamorphosis[61]. Those research indicated that TIMPs were involved in a variety of tumorigenic pathways and are expected to use this gene family as a link in immunotherapy to specifically affect the efficacy of immunotherapy [13].
Nowadays, synthetic MMP inhibitors have been developed and evaluated for the treatment of a range of tumors, and their efficacy has aroused our interest, including hydroxamate-based inhibitors, non-hydroxamate MMP inhibitors, new generation of hydroxamate-based MMP inhibitors, targeting alternative binding sites, antibody-based therapeutics, and endogenous inhibitors of MMP function[22]. Notably, being the first MMP inhibitors to be produced, hydroxamate-based inhibitors primarily inhibit many MMPs, like MMP-1, MMP-2, MMP-7, and MMP-9, presenting antitumor effects of human colorectal cancer, ovarian cancer, melanoma, and hemangioma in animal models[62]. New generation of hydroxamate-based MMP inhibitors was employed to treat osteoarthritis and rheumatoid arthritis[63, 64]. The precise physiology and biology of MMPs, as well as the mechanisms of their evolution remain unclear, and it can exert negative consequences like musculoskeletal toxicity, hence there is still a long way to go for the development of MMP synthesis inhibitors[22, 62].
In conclusion, because TIMP1 has pro- and antitumoral effects, its role in cancer is still controversial[22]. As mentioned above, TIMP2 may prevent cell proliferation, angiogenesis, and tumor development. Even so, the disease-related expression profile and prognosis trend of TIMP2 are quite heterogeneous, which appears to be dependent on the origin tissues of the cancers[30]. TIMP3 is considered as a tumor suppressor[48]. Few studies have been conducted on TIMP4, so its role in tumors cannot be adequately and exhaustively summarized.