Anoikis is a specific type of cell death that has received much attention in recent years[20]. Several studies have elucidated the various mechanisms employed by cancer cells to eliminate anoikis and promote their metastasis and invasion. Therapeutic approaches targeting anoikis have been initiated in some cancers[21]. In studies on CRC, genes such as CPT1A, IF1 and KLF5 have been found to be involved in anoikis resistance[14, 16, 17]. However, an anoikis signature in CRC has not been systematically identified. Therefore, our study explored the role of anoikis in the development and metastasis of CRC through bioinformatics.
In the study, the prognosis of cluster A was worse than that of cluster B, including in patients who also received chemotherapy. This suggests that there is heterogeneity in the role of anoikis in CRC patients, which may benefit patients receiving chemotherapy. Anoikis resistance is an important feature of tumor metastasis. We noticed a significant increase in the proportion of stage III or IV patients in cluster A compared with cluster B. This suggests that there may be resistance to anoikis in cluster A, which could make cluster A patients more likely to develop lymph node or distant metastases. Cluster B, conversely, showed the opposite possibility. This distinction indicates that anoikis has distinct modes of action in CRC. We considered cluster A to be an anoikis-resistant cluster and cluster B an anoikis-activated cluster.
To explore this difference, we did a differential gene analysis of the two clusters. Intriguingly, some genes related to tumor development and progression showed a decreased expression in cluster B and increased expression in cluster A. In previous reports, FNDC1 and SFRP4 were closely linked to the development of epithelial-mesenchymal transition (EMT), which represents the ability to acquire migration[22, 23]. Further, knockdown of FNDC1 was found to inhibit proliferation, invasion and migration of gastric cancer cells, and by modulating the Wnt/β-catenin signaling pathway, FNDC1 may facilitate the EMT of gastric cancer cells[22]. Not coincidentally, SFRP4 expression was also found to be proportional to tumor invasion in gastric cancer, but the exact mechanism has not been elucidated[23]. In addition, Bin et al. found by bioinformatics methods that a high THBS2 expression was associated with a shortened survival, and this alteration was associated with the PI3K-AKT pathway and ECM. The authors thus considered it a potential biomarker of CRC[24]. Jie et al. further found that the knockdown of OLFM4 was able to promote metastasis of gastric cancer cells by activating the NF-κB/IL-8 axis[25]. Such an altered gene expression may promote the activation of anoikis in cluster B and prevent tumor metastasis.
We also noticed differences in functional enrichment. We are concerned that many ECM-related pathways are enriched. The normal cell survival is signaled by cell adhesion to ECM components as well as soluble growth factors and cytokines, while inappropriate ECM attachment or loss of cellular machinery anchoring is a key mechanism causing anoikis[26]. Failure to execute the anoikis program may lead to adnexal cells at the site of initial contact between matrix proteins and cell proliferation at ectopic sites where the stromal proteins are different from the stromal proteins[27]. This deregulation is an important hallmark of metastatic cancer cells. Our findings suggest that this resistance may emerge in CRC as a noteworthy research direction.
In cluster A, we discovered the increased expression of a number of pathways that have been reported in cancer metastasis studies. Jackstadt et al. found that the NOTCH signaling pathway promotes tumor metastasis by creating a microenvironment that attracts neutrophils[28]. The relationship between NOTCH signaling and anoikis has been reported in cervical tumors and prostate cancer. In cervical tumors, the activation of NOTCH signaling has been found to promote tumor metastasis by activating the PKB/Akt pathway and generating resistance to anoikis[29]. In prostate cancer, increased NOTCH signaling can inhibit anoikis in prostate epithelial cells by enhancing NF-κB activity[30].
Moreover, the MAPK signaling pathway has been implicated in CRC metastasis[31]. This pathway is crucial for the regulation of cell-cell and cell-matrix contacts, and its disruption in the initiation of the anoikis program results in the death of non-normal cells, such as cancer cells[8]. The TGF-β pathway is an important pathway in the development of metastasis in CRC and has been reported to be linked to the loss of SMAD4, a transcription factor in TGF-β superfamily signaling that promotes tumor growth[32]. In previous reports, its association with anoikis resistance has also been suggested. Zou et al. reported that INHBB, a TGF family protein inhibits anoikis resistance and metastasis in nasopharyngeal carcinoma cells through attenuating the action of the TGF-β pathway[33]. In some CRC cases, TGF-β signaling, by increasing anoikis resistance, may enhance the ability of CRC cells to invade and colonize a second site[34].
In the study, the expression of the TGF-β, MAPK and NOTCH signaling pathways was significantly increased in cluster A, further suggesting the presence of anoikis resistance in cluster A patients. These pathways were indeed reported to be associated with CRC metastasis in previous studies. Whether or not these pathways mediate anoikis resistance in CRC needs to be further investigated.
Changes in the TME are closely related to tumor metastasis[35]. In our immune analysis, anoikis resistance may be associated with immune activation. We also focused on the increased infiltration of MDSCs, macrophages and regulatory T cells in cluster A. In a previous report, macrophages in neoplasms were shown to drive angiogenesis and promote tumor cell migration and invasion [36]. In HCC, M2 macrophages promote HCC cell migration and EMT via the TLR4/STAT3 signaling pathway[37]. In breast cancer, macrophage abundance positively correlates with EMT marker[38]. Such a relationship has also been reported in CRC. Tumor-associated macrophages (TAMs) can induce EMT by regulating the JAK2/STAT3/miR-506-3p/FoxQ1 axis to enhance CRC migration and invasion[39]. In addition, MDSCs not only suppress the immune system during tumor progression, but they also accelerate tumor growth, metastasis, and angiogenesis through VEGF[40]. Tregs show interaction with MDSCs in cancer, and the activation of Tregs by MDSCs is mainly caused by cytokines, such as IL-10 and TGF-β, which are also associated with the induction of MDSCs[41]. In CRC, the presence of a large number of Tregs reportedly predicts a poor prognosis [42]. In our study, in the immune-activated microenvironment of cluster A, a large number of MDSCs, M2s and other immune cells were highly expressed, and such changes may be involved in the formation of anoikis resistance and promote tumor survival and metastasis.
CRC gene mutation research has been hot recently. The differences between the high- and low-risk groups may be related to the anoikis-related genes mutation. We focused on the BRAF gene mutation group, which had a higher risk score than the BRAF wild group. BRAF mutations can affect the MEKERK pathway, allowing cancer cells to survive in the absence of integrin-involved survival signals[27, 43]. This relationship has been previously demonstrated in melanoma. Patankar et al. found that BRAF mutations induce anoikis resistance in CRC, suggesting that this mechanism is likely generated by activation of the RAS-RAF-ERK pathway[44].
Finally, the prognostic significance of the model can be further strengthened after we further integrate the clinical features, and the predicted prognostic nomogram can effectively achieve individualized risk assessment.
Admittedly, our study has several limitations. First, our analysis is based on a publicly accessible database and needs independent prognostic cohort validation. Second, more tests are required to verify the involvement of anoikis in CRC.