Firstly, the current section of our review will focus on total CD44 involvement in chemoresistance, since total CD44-mediated signaling may involve both standard and/or variant exon containing isoforms. Onwards, specific variant exons of CD44 will be discussed.

Overall, a significant negative effect of increased total CD44 expression on chemotherapy treatment outcome was observed across several types of cancers, including breast, colorectal and head and neck cancers measured typically through overall survival (OS) and recurrence-free survival (RFS) values, as well as through response to chemotherapy (Wu et al., 2024). Notably, several studies have identified no correlation or even a positive correlation with response to chemotherapy treatment outcome, which underlines the ambiguity of CD44’s relationship with chemotherapy outcomes (Wu et al., 2024). Such differences may be due to the variation of CD44 variant isoforms amongst different cancer types and the difference in activated survival pathways. Varying CD44 isoform/variant exon detection methods may also contribute to these differences.

In addition, the association between total CD44 overexpression/knockdown and chemoresistance in vivo has been studied (Table 2). Research indicates that CD44 knockdown (CD44^kd) in prostate cancer xenografts significantly reduces tumor growth rate after continuous docetaxel treatment compared with control cells, suggesting that the combination of chemotherapy with CD44^kd can lead to a beneficial chemotherapy outcome (Hao et al., 2012).

Literature analysis demonstrated a predominantly negative relationship between total CD44 expression levels and the clinical outcome of patients. Moreover, CD44^kd cancer xenografts in vivo significantly reduce tumor growth rate after continuous chemotherapy administration. Thus, total CD44, despite the presence of both standard and variant isoforms, seems to be involved in chemotherapy resistance.

The underlying mechanisms of total CD44 in chemotherapy resistance are quite diverse, possibly due to its combination of several variant exon-containing isoforms and the standard isoform (Table 3). It has been shown to influence key cell death signaling pathways, including apoptosis, ferroptosis and autophagy.

It was shown that CD44 is able to form a complex with receptor tyrosine-protein kinases 2 (ERBB2) (Ghatak et al., 2005). For instance, HA oligomers, inhibitors of HA interaction with CD44, were shown to prevent the assembly of phosphorylated ERBB2, CD44, ezrin, Hsp90/Cdc37 and p110a into a complex, which is then able to exert ERBB2 associated PI3K/Akt signaling pathway, promoting apoptosis resistance (Ghatak et al., 2005; Figure 3A). Such an interaction demonstrates the involvement of CD44 in growth factor receptor signaling promotion.

Moreover, research on CD44 influence on cell death signaling pathways uncovered its involvement in the Hippo pathway. It was demonstrated that upon HA binding to CD44, CD44 clustering on the cellular membrane is initiated, stimulating the interaction of its intracellular domain with polarity-regulating kinase (PAR1b) (Ooki et al., 2019). Notably, it has been demonstrated that in normal tissues with increasing cell densities, CD44 is activated by high molecular weight (HMW)-HA, thereby initiating PAR1b phosphorylation process of mammalian STE20 like kinase 1 and 2 (MST1/2) and activation of Yes associated protein (YAP). Such a process results in the inhibition of cell growth. Similarly, merlin/neurofibromin-2 (Mer/NF2) also acts as a downstream effector, transferring signals from HA-CD44 activation. CD44 activation by HMW-HA reduces Mer phosphorylation, leading to its activation. Activated Mer has been shown to inhibit PI3K/Akt signaling (Ooki and Hatakeyama, 2020). Overall, in parallel with PAR1b, Mer acts as downstream effector molecules of the HA/CD44/Hippo pathway, modulating its activity. Since the Hippo pathway is an essential survival-associated signaling pathway, its’ inactivation promotes cell proliferation and decreases apoptosis, contributing to tumor initiation, progression and chemoresistance (Figure 3B).

Another pathway utilized by tumor cells to prevent apoptosis is signaling through the CD44/CD147/EGFR complex. On one hand, it was demonstrated that the knockdown of total CD44 leads to decreased EGFR activity levels, pointing to their functional linkage with one another (Grass et al., 2013). On the other hand, CD147 was shown to promote HA synthesis, thereby acting on CD44 activity levels through ligand interactions (Grass et al., 2013). The authors further demonstrated that the CD44/CD147/EGFR complex forms and promotes ERK activation through enhanced CD44-HA signaling in lipid rafts (Grass et al., 2013). While the impact of ERK activation on apoptosis was not directly analyzed, EGFR-Ras-ERK signaling is known to inhibit apoptosis and promote cell survival, thereby implying yet another case of CD44 apoptosis regulation through the interaction with cell membrane receptors (Figure 3C; Ullah et al., 2022).

Apart from apoptosis resistance, total CD44 may be involved in ferroptosis resistance. Müller et al. described a novel mechanism involving hyaluronan-bound iron transfer into the cell via CD44, influencing gene expression regulation (Müller et al., 2020; Figure 3D). The authors demonstrated that LMW-HA bound by iron is internalized intracellularly in complex with CD44 by endocytosis. Similar to transferrin-bound iron, CD44-HA-bound iron is further released into the cytoplasm upon acidification of the endosome and can then be transferred into specific cellular compartments. In particular, CD44-HA-bound iron increases the nuclear iron pool and reduces the histone mark H3K9me2 in an iron-dependent manner (Müller et al., 2020). This leads to the upregulation of genes, including CD44 itself. While no current studies suggest that CD44-mediated iron flux directly contributes to chemoresistance, a potential link may exist. Additionally, the impact of different CD44 isoforms on the CD44-HA-iron feedback loop remains unclear.

Total CD44 is additionally known to regulate autophagy in cancer cells. For instance, autophagy was shown to promote a CD44^high/CD24^low phenotype in breast cancer stem cells (CSCs) – such a cell expression profile was identified in a smaller portion of cells upon knockdown of several autophagy initiating proteins (ATG8/LC3B and ATG12) (Cufí et al., 2011). Moreover, the treatment of CD44^high/CD24^low cells with an autophagy inhibitor chloroquine (CQ) was shown to shift the cancer cell phenotype to the opposite (CD44^low/CD24^high) (Cufí et al., 2011). Additionally, the CD44 ligand osteopontin (OPN) has been implicated in autophagy induction. In vascular smooth muscle cells (SMCs), OPN was shown to trigger autophagosome formation (Kariya and Kariya, 2022). This process was inhibited by an integrin (ITG) inhibitor (RGD peptide) or anti-CD44 antibody, highlighting the importance of CD44 and RDG motif-containing integrins in autophagy initiation (Figure 3E). In another study, OPN activation was shown to stimulate NF-κB, ERK, and STAT3 signaling pathways (Figure 3F; Zheng et al., 2012). Thus, literature suggests that total CD44 and its ligands are linked with autophagy initiation and the stemness properties of tumor cells, thereby promoting resistance to administered therapy.

Overall, the mechanisms responsible for total CD44 mediation of cell death pathways are diverse. They include the influence of apoptosis resistance through ERBB2 complex, CD147 and CD44 interaction with LMW-HA. Currently, no research highlights that iron flux mediated by CD44 significantly contributes to chemoresistance, but there may be an association. Whether different CD44 isoforms alter their expression patterns within the CD44-HA bound iron positive feedback loop remains unclear. Further studies are required to clarify these possible associations and their relationship with chemoresistance. Lastly, total CD44 was shown to promote autophagy and chemoresistance in several cancers through OPN-CD44-ITG-p38MAPK and OPN-CD44-NF-κB signaling. Thus, targeting both cell death pathways and total CD44 signaling may offer a promising approach to overcoming chemoresistance in various cancers. However, it should be mentioned that additional research comparing the effect of standard and variant isoforms in mediating cell death signaling may be necessary for further precision in targeting chemoresistant cells.

Several approaches targeting total CD44 (including variant and standard isoforms) for chemotherapy resistance alleviation have been developed and analyzed in vivo (Table 4). Some approaches for CD44-exerted chemoresistance treatment entail using CD44-targeting gene therapies, delivery systems and cell therapies. Regarding gene therapy strategies, complementary DNA (cDNA) vaccine to CD44 variant isoforms, but not CD44s, demonstrated decreased tumor growth, aggressiveness and metastasis in breast cancer xenografts (Wallach-Dayan et al., 2008). It was identified that the cDNA vaccine induced the production of antibodies recognizing CD44 variant isoforms (Wallach-Dayan et al., 2008). In addition, several studies revealed the beneficial effect of CD44-targeting with micro-RNA (miRNA). MiR-199a overexpression in CD44⁺ cancer-initiating cells (CICs) in ovarian cancer xenografts significantly decreased tumor volume (Cheng et al., 2012). Additionally, it was identified that a more significant percentage of apoptotic cells is formed with miR-199a overexpression. Similar effects were observed with the overexpression of miR-24a and miR-34a in gastric and esophageal cancer xenografts (Jang et al., 2016; Zuo et al., 2018). Overall, the data surrounding gene therapy approaches seems promising regarding cancer patient treatment, but they should be cautiously studied since they can carry the risk of off-target effects.

Moreover, CD44 variant isoforms can be helpful for tumor cell targeting (cell therapies) and chemotherapy delivery due to their overexpression in tumor tissue. Anti-CD44 variant isoforms micelles, incorporating cisplatin, have shown success in decreasing the tumor volume of oral cancer xenografts by more than 50% (Wang et al. (2016a)). In addition, CD44-targeted and pH-sensitive doxorubicin-loaded micelles have exerted selective cytotoxic effects in breast cancer xenografts, resulting in apoptosis induction (Yang et al., 2021). A recently developed method for tumor cell targeting is therapy with chimeric antigen receptor (CAR) T cells, successfully applied for CD44⁺ tumor cell elimination. For instance, CD44⁺ hepatocellular carcinoma xenografts treated with CD44-CAR T cells demonstrated a significant decrease in tumor volume in comparison with mock T and normal T cells; CD44 tumor volumes were unchanged in comparison with CD44-CAR T, mock T and normal T cells (Wang et al., 2020).

Even though preclinical research data suggest that total CD44 play a significant role in the chemoresistance of cancer patients, there is a limited amount of conducted clinical studies regarding total CD44 targeted therapies (Table 5). They include therapeutic approaches such as using antibodies/peptides or pharmacological inhibitors.

Anti-total-CD44 targeting approaches with modest/insufficient effect have been noted (NCT01358903): an anti-CD44 antibody RO5429083 (alternatively named RG7356) was tested in metastatic and/or locally advanced CD44-expressing malignant solid tumors (Menke-van der Houven van Oordt et al., 2016). The developed antibodies demonstrated an acceptable safety profile, however, clinical efficacy was identified to be modest–the best response to chemotherapy was stabilization of disease progression identified in 21% of patients. Some promising clinical trials have also been conducted (NCT00083928 and NCT01553851). A6 peptide, a short amino acid sequence targeting the HA-binding domain of CD44, performed well in increasing the time to progression of patients with ovarian cancer: half of the patients with asymptomatic biochemical recurrence experienced periods of stable disease for at least 4 cycles of administered A6 peptide therapy (Ghamande et al., 2008). Moreover, trametinib (conversely named GSK1120212) was shown to decrease Ras/MEK/ERK pathway activation and CD44 expression, resulting in a beneficial clinical response in patients (the effect was seen in 11 out of 17 patients), a decrease in tumor volume and tumor downstaging (Figure 4; Uppaluri et al., 2017).

CD44v3 seems to be the least studied variant exon out of the group of clinically relevant exons (Table 6). Concerning the correlation of expression levels with clinical outcomes of patients, it was demonstrated that high protein expression levels of CD44v3 exert chemoradiotherapy resistance in a study with nasopharyngeal patients (Sagawa et al., 2016). There seem to be no studies examining the association between CD44v3-containing isoforms overexpression/knockdown and chemoresistance in vivo. Nevertheless, the biological effect of CD44v3 has been studied in vitro.

The biological effects exerted by variant exons of CD44 with significant clinical association concerning cell death pathways are summarized in Table 3. They will be covered in detail in the upcoming sections of our review.

The study of a particular exon’s functional role is quite tricky. Such methods as overexpression/knockdown of particular CD44 isoforms, which typically contain more than one variant exon, are widely applied. Indeed, we are not the first to review the involvement of CD44 variant exons in chemoresistance induction through apoptosis regulation (Wang et al., 2018). However, Wang et al. primarily focus on variant exon v6. In our review, we outline what is known to date about all clinically relevant variant exons regarding cell death mechanisms, including apoptosis, ferroptosis and autophagy.

Firstly, CD44 has been shown to bind some metalloproteinases (MMPs), a family of endopeptidases that hydrolyze components of the extracellular matrix (Hassn Mesrati et al., 2021). Specifically, CD44v3 containing isoforms were identified to recruit the active form of MMP-7 and the precursor of HB-endothelial growth factor (HB-EGF) at its extracellular domain. This is possible due to HB-EGF binding of HS-rich sites in variant exon v3 of CD44 (Yu et al., 2002). Such an interaction allows the presentation of mature HB-EGF to membrane receptors, such as ERBB4, and thus the promotion of cell survival (Figure 5A; Yu et al., 2002). The mechanism through which CD44 forms complexes at the cellular membrane with additional receptors is quite often utilized by cancer cells and seems to be successful in the promotion of cell death resistance.

Moreover, CD44v3-containing isoforms were shown to promote ovarian tumor growth through ERBB2 signaling. It was identified that the ICD of CD44v3-containing isoforms interacts with adaptor proteins Vav2 and growth factor receptor bound protein 2 (Grb2) (Bourguignon et al., 2001), thus promoting coupling of ERBB2 and CD44v3-containing isoforms to Ras and the promotion of its signaling (Bourguignon et al., 2001; Figure 5B). This interaction is similar to that of total CD44 and ERBB2, but the mechanisms of apoptosis promotion vary. This points to a nonuniform response of tumor cells from different cancers upon interaction of CD44 with the same receptor.

Additionally, CD44v3 was shown to promote apoptosis resistance through epigenetic regulatory mechanisms. For instance, in CSCs with a CD44v3^high/ALDH1^high phenotype, master transcriptional factors Oct4, Sox2 and Nanog were detected to be overexpressed at the mRNA and protein levels (Bourguignon et al., 2012). Interestingly, upon activation of CD44v3-containing isoforms by HA in such cells, CD44v3-associated Oct4, Sox2 and Nanog physical complex formation and dislocation of Oct4/Sox2/Nanog to the nucleus was observed (Bourguignon et al., 2012). Upon nuclear localization, Oct4/Sox2/Nanog was identified to bind the miR-302 cluster promoter region, enhancing the production of miR-302a and miR-302b expression. SiRNA-mediated knockdown of these factors blocked HA-mediated Oct4/Sox2/Nanog binding to the miR-302 cluster promoter and decreased HA-induced antiapoptotic IAP protein expression, promoting chemosensitivity to cisplatin (Bourguignon et al., 2012). Thus, these finding indicate the ability of CD44v3 to utilize regulatory mechanisms not only at the protein level, but also epigenetically.

Further investigations into the role of CD44v3 in apoptosis regulation unveiled that the knockdown of CD44v3-containing isoforms mediates the reduction of PI3K, pAKT, pERK, pSTAT3 and Bcl2 protein levels, triggering apoptotic signaling and G0/G1 phase arrest in bladder cancer cells (Anand et al., 2019). Combinational treatment of cisplatin and doxorubicin with 4-methylumbelliferone (4-MU), an HA synthesis inhibitor, significantly decreased cell viability compared to cisplatin or doxorubicin alone, suggesting that the HA/CD44v3 axis is of importance in apoptotic signaling regulation (Figure 5D; Anand et al., 2019).

As mentioned previously, CD44v3 encodes a heparan sulphate (HS) side-chain attachment motif, allowing HS-bound CD44v3 to bind growth factors including hepatocyte growth factor (HGF) and to present them to a number of cell membrane receptors including the mesenchymal epithelial transition factor receptor (c-Met) (van der Voort et al., 1999). A significant pathway of c-Met signaling is the PI3K/Akt signaling axis primarily responsible for the cell survival response: the p85 subunit of PI3K can bind either directly to c-Met or indirectly through GAB1, which in turn promotes anti-apoptotic signaling through AKT (Xiao et al., 2001). Thus, these findings underline the importance of HS motifs and their utilization by CD44v3-containing isoforms to promote antiapoptotic signaling.

Overall, CD44v3-containing isoforms contribute to apoptosis resistance through several mechanisms, including the c-Met and ERBB2 receptors, epigenetic regulation employing miR-302 cluster upregulation and MMPs. Further research on these pathways will facilitate the development of targeted therapies to overcome chemoresistance and improve cancer treatment outcomes.

Despite the relatively large number of molecular mechanisms of resistance in which CD44v3 partakes, there seem to be practically no antibodies or clinical trials aimed at targeting CD44v3. A novel monoclonal antibody, C44Mab-6, was recently established (Suzuki et al., 2023). One advantage of such antibodies is the area of recognition of CD44v3-containing isoforms. For instance, compared with previously established anti-CD44v3 antibodies (clone 3G5), C44Mab-6 recognizes a peptide sequence excluding an HS-modified sequence in the CD44 variant-3-encoded region, indicating that C44Mab-6 antibodies recognition of CD44v3 is not influenced by HS modifications (Suzuki et al., 2023). This may be useful for CD44v3 targeting, increasing the selectivity of such antibodies. The authors mention that further testing of C44Mab-6 in vivo is reckoned.

Apart from the development of the antibodies mentioned above, it does not appear that other antibodies have been synthesized and tested in clinical settings. Thus, further research is necessary to determine the relevance of CD44v3 inhibition, considering the small number of studies regarding the analysis of its clinical relevance.

One of the most widely and thoroughly studied variant exons concerning the clinical outcome of patients and biological effect is CD44v6 (Table 6). Several studies unveiled the prognostic significance of CD44v6 in nasopharyngeal, esophageal (in combination with c-Met expression) and tongue cancer (Hara et al., 2019; Sagawa et al., 2016; Yanamoto et al., 2014). High protein expression levels of CD44v6 significantly correlated with poor disease-specific survival (DSS) and OS of patients treated with cisplatin/carboplatin-based chemotherapy, as well as with tumor recurrence after chemotherapy treatment in colorectal cancer patients (Lv et al., 2016).

In addition, the association between CD44v6 overexpression/knockdown and chemoresistance in vivo has been studied (Table 2). Research indicates that CD44v6^kd in prostate and colon cancer xenografts demonstrated a significant decrease in tumor growth and practically complete impalpability by the end of docetaxel administration and in folinic acid, 5-FU and oxaliplatin (FOLFOX)-resistant cells (Ni et al., 2020; Ghatak et al., 2021).

Overall, the implemented literature analysis, although not comprehensive, pointed to a relationship between variant exon CD44v6 high expression levels and worse chemotherapy treatment outcomes in cancer patients. Cancer xenografts confirmed the negative relationship of CD44v6 expression level with worse chemotherapy efficacy in vivo. The results demonstrate that CD44v6 may be involved in chemotherapy resistance, and further research into its molecular mechanisms is needed.

The ongoing investigation into the role of CD44 variant exon v6 in cellular death pathways across various cancer types may not yet provide a comprehensive understanding for all cancers. To date, CD44v6 is the most studied variant exon out of the other clinically variant exons. It has been shown to influence apoptosis and autophagy.

One of the pioneering studies on CD44v6 involvement in apoptosis revealed that CD44 exon v6 containing isoforms exhibit antiapoptotic properties through colocalizing, interacting with and blocking FAS trimerization (Figure 6A; Mielgo et al., 2006). Notably, cells, constitutively expressing FASR and being sensitive to apoptosis with the addition of FASL or FAS-crosslinking antibodies, become resistant to apoptosis with the transfection of CD44 variant isoforms. It was demonstrated that blocking the CD44v6 exon with anti-CD44v6 antibodies (clone VFF18) in cancer cells results in their sensitization to apoptosis, thereby underscoring CD44v6’s role in the promotion of antiapoptotic signaling. The authors further propose that CD44 variant isoforms may interact with the pre-ligand assembly domain of FASR and prevent its trimerization, which is necessary for FASL binding (Mielgo et al., 2006). Thus, CD44v6-containing isoforms employ regulatory mechanisms acting directly on the extrinsic apoptotic signaling pathway.

Moreover, research indicates that CD44v6 interacts with the mesenchymal epithelial transition factor receptor c-Met and inhibits apoptosis through two primary mechanisms: 1) presentation of HGF to c-Met or 2) by direct interaction of c-Met or HGF with the variant exon domain of CD44v6 (Figure 6B). Based on current studies, the function of CD44v6 in c-Met activation is of dual nature. Firstly, the ectodomain of CD44v6-containing isoforms is necessary for c-Met activation through the binding and presentation of HGF. Secondly, the cytoplasmic domain of CD44v6-containing isoforms is linked to ERM proteins and the cytoskeleton and is required for RAS activation (Hasenauer et al., 2013). Overall, CD44v6-c-Met interaction promotes apoptosis through PI3K/Akt and mammalian target of rapamycin (mTOR) pathways (Jung et al., 2011). Notably, studies indicate that upon the addition and interaction of HGF with CD44v6 expressing cancer cells, there is an increase in the number of CD44-HGF-bound dimers and in the diffusion coefficient in the plasma membrane of such complexes (Tannoo et al., 2024). The latter process is favorable for rapid interaction with and activation of c-Met, resulting in the formation of a CD44v6-c-Met tertiary complex (Tannoo et al., 2024). Therefore, tumor cells expressing CD44v6-containing isoforms utilize variant exon v6 for binding of growth factors and their presentation to cell membrane receptors (or even directly interacting with them), promoting anti-apoptotic signaling initiation.

Moreover, the formation of a CD44v6/transglutaminase 2 (TG2)/ERK1/2 protein complex was shown to promote cancer cell growth and resistance to apoptosis (Chen et al., 2023). It was identified that TG2 knockdown leads to decreased levels of CD44v6 and reduced ERK1/2 activity and expression (Chen et al., 2023). Furthermore, TG2 and CD44v6 were found to form complexes with ERK1/2, as well as with each other. In the interaction involving TG2 and CD44v6-containing isoforms, the cytoplasmic domain of CD44 plays a critical role in maintaining the stability of the complex (Chen et al., 2023). Importantly, CD44v6-knockdown xenografts displayed a significant decrease in tumor growth, accompanied by reduced CD44v6 and ERK1/2 levels, thereby utilizing yet another cooperative mechanism with binding partners for anti-apoptotic signaling promotion (Figure 6C; Chen et al., 2023).

Furthermore, overexpression of CD44 isoforms containing CD44v6 (CD44v6-overexpressing cells) demonstrated an increase in resistance to 5-FU in colorectal cancer cells (Lv et al., 2016). An increase in BECN1 expression under treatment by 5-FU in CD44v6-overexpressing cells was identified, and treatment of CD44v6-overexpressing cells with 5-FU and an autophagy inhibitor quinacrine (QC) resulted in an increase of CD44v6-overexpressing cell’s sensitivity to the combination of 5-FU and QC, indicating that CD44v6 promotes autophagy resistance. In another study, the knockdown of CD44v6 containing isoforms in colorectal cancer cells resulted in a significant decrease in BECN1 levels, further validating the results obtained in the previously mentioned study (Wang et al., 2019; Figure 6D).

Thus, the role of CD44v6 in apoptosis resistance, autophagy promotion, and consequent chemoresistance attenuation is complex, and it is involved in modulating several signaling pathways and interactions. CD44v6-containing isoforms contribute to apoptosis resistance through mechanisms involved in both direct apoptosis regulation via FASR and indirect apoptosis influence through the c-Met receptor. Additionally, CD44v6-containing isoforms were shown to promote autophagy in several cancers. These interactions underline the complexity of CD44’s functions responsible for cancer progression and resistance to treatment. The development of targeted therapeutic drugs for overcoming chemoresistance greatly depends on understanding CD44 variant exon-containing isoforms manipulation of cell survival signaling pathways.

Given the seemingly promising role of CD44v6-containing isoforms in cancer chemoresistance demonstrated in preclinical studies, several approaches targeting CD44v6 for chemotherapy resistance alleviation have been developed and analyzed in vivo (Table 4). These strategies aim to disrupt CD44-mediated signaling, enhance cell death induction and, as a result, overcome chemoresistance.

Some approaches for CD44v6-exerted chemoresistance treatment entail the use of anti-CD44v6 antibodies/peptides or pharmacological inhibitors. For instance, CD44v6 targeting peptides Hv6pep, developed with the inclusion of 3 amino acids necessary for v6 interaction with coreceptors, induced a significant tumor growth reduction upon xenograft injection. Notably, the anti-CD44v6 peptide was more effective in tumor growth inhibition than inhibitors of CD44v6 interactions with coreceptors c-Met and vascular endothelial growth factor receptor 2 (VEGFR-2) (Matzke-Ogi et al., 2016). A recently developed method for tumor cell targeting is therapy with chimeric antigen receptor (CAR) T cells, successfully applied for CD44v6 tumor cell elimination. For instance, the developed CD44v6-CAR T cells demonstrated antitumor effects and significantly prolonged survival (median survival of 27 and 37 days in CD19-CAR and CD44v6-CAR treated groups) of mice with ovarian cancer tumors (Porcellini et al., 2020). Thus, CD44v6-expressing isoforms are helpful for selective and direct tumor cell annihilation and precise drug delivery to chemoresistant cells.

Even though preclinical research data and several case reports suggest that CD44v6-containing isoforms play a significant role in the chemoresistance of cancer patients, there is a limited amount of conducted clinical studies regarding CD44v6-targeted therapies (Table 5). Cases of insufficiently effective anti-CD44v6 targeting approaches have been noted (NCT01358903, NCT01358903 and NCT02254018).

An anti-CD44v6 antibody, bivatuzumab, in combination with mertansine, was tested in studies targeting recurrent or metastatic breast cancer and advanced head and neck carcinoma (Figure 7; Koppe et al., 2004; Riechelmann et al., 2008; Ishimoto et al., 2011). In the cases of both metastatic breast cancer and advanced head and neck carcinoma, bivatuzumab was shown to target CD44v6, although not only on tumorous tissue, but additionally on skin keratinocytes - cells strongly expressing CD44v6. In the head and neck carcinoma study, one patient developed stable disease during the treatment phase, but a patient’s drug-related severe skin toxicity caused the study termination due to their death. Lastly, phase I/II clinical studies aimed at targeting CD44v6 with CAR T cells (NCT04430595, NCT04427449 and NCT04097301) and with pharmacological inhibitor AMC303 (NCT03009214) have demonstrated exemplary safety, and further analysis with respect to clinical effect is expected (Figure 7).

Another frequently studied variant exon is CD44v9, which was shown to be associated with poor RFS and DSS of the upper urinary tract and urothelial cancer patients undergoing cisplatin-based chemotherapy, as well as non-chemoradioselected advanced head and neck cancer patients (Aso et al., 2015; Hagiwara et al., 2016; Hagiwara et al. 2018; Table 6). Moreover, the association between CD44v9-containing isoforms overexpression/knockdown and chemoresistance in vivo has been studied (Table 2). Chemoresistance elevation in CD44v9-overexpressing gastric cancer xenografts was averted in the 5-FU and sulfasalazine (SAS)-treated groups, demonstrating tumor volume shrinkage when compared to both the control group and 5-FU group on days 21 and 28 (Miyoshi et al., 2018).

Overall, the implemented literature analysis pointed to a relationship between variant exon CD44v9 high expression levels and worse chemotherapy treatment outcomes of cancer patients. Cancer xenografts confirmed the negative relationship of variant exon v9 expression level with worse chemotherapy efficacy in vivo. The results demonstrate that CD44v9, in addition to CD44v6, may be important in chemotherapy resistance occurrence.

To date, CD44v9, alongside CD44v6, is a frequently studied variant exon compared to other clinically variant exons. It has been shown to influence apoptosis and, to a greater extent, ferroptosis in cancer cells (Table 3).

For instance, CD44v9-containing isoforms transfected to cancer cells demonstrated that CD44v9-overexpressing cells were significantly more resistant to apoptosis than CD44v3 and CD44s (Mielgo et al., 2006). However, blocking of CD44v9-containing isoforms with anti-CD44v9 antibodies (clone FW11.24) demonstrated their inability to restore the potential of cells to undergo apoptosis (Figure 6A; Mielgo et al., 2006). Therefore, we conclude that the noted effects may be explained by several reasons: 1) the antibodies used in the study do not properly bind to and interact with CD44v9, therefore apoptosis restoration was not detected and 2) this mechanism may be more supportive, rather than being a major one.

Research conducted by Ishimoto et al. elucidated the interplay between CD44v9-containing isoforms and cystine-glutamate transporter xCT, a key player in glutathione (GSH) synthesis, reactive oxygen species (ROS) inactivation and ferroptosis regulation (Koppula et al., 2021). Their findings indicate that CD44 variant exons v8-v10 in gastrointestinal cancer (or CD44v9-containing isoforms in other types of cancers) interact with and stabilize the cystine transporter xCT, preventing it from being internalized and degraded as well as enhancing its capacity for downstream GSH synthesis and defense against ROS (Figure 8; Ishimoto et al., 2011). Interestingly, with the introduction of a S301A mutation containing consensus motifs for an N-linked glycosylation site in the variant region of CD44 (CD44v8-v10), CD44 variant isoforms failed to interact with xCT, thus underlining the importance of posttranslational modifications for CD44v9/membrane proteins interactions (Ishimoto et al., 2011). The precise molecular mechanisms underlying this interaction and stabilization are still under investigation but are thought to involve direct protein-protein interactions and possibly glycosylation-dependent processes.

Additional stability and degradation escape of CD44v9-xCT complex is carried out through interaction with ubiquitin thioesterase/otubain-1 (OTUB1), a deubiquitinating enzyme (DUB) that plays a vital role in regulating protein stability by removing ubiquitin molecules from target proteins, thereby preventing their degradation by the proteasome and as a result mediating ferroptosis (Liu et al., 2019; Figure 4A). OTUB1 has been shown to directly interact with and stabilize the xCT transporter: knockdown of OTUB1 led to growth suppression of mice tumor xenografts and reduced activation of ferroptosis (Liu et al., 2019). Interestingly, CD44 demonstrated the ability to stabilize xCT and OTUB1 interaction in an OTUB1-dependent manner. Moreover, the N-terminal domain of xCT is required for interacting with OTUB1, whereas the C-terminal domain of xCT is critical for binding CD44 (Liu et al., 2019). Although the direct interaction of CD44 and OTUB1 is much lower in comparison to OTUB1-xCT interaction, nevertheless, this three-protein complex is thought to stabilize xCT more effectively, possibly further enhancing chemoresistance.

Another player involved in CD44v9-xCT complex stabilization is mucin 1 (MUC1) – a transmembrane glycoprotein commonly overexpressed in a plethora of cancers including lung, colon, breast, pancreatic and ovarian cancers (Chen et al., 2021; Figure 4A). Hasegawa et al. uncovered the interaction between the oncogenic MUC1 C-terminal subunit (MUC1-C) and the CD44-xCT complex: it was demonstrated that MUC1-C extracellularly interacts with and stabilizes the xCT transporter and additionally interacts intracellularly with CD44 ICD (Hasegawa et al., 2016). It was demonstrated that treatment of MUC1-C^high cells with ferroptosis inducer/xCT inhibitor erastin was ineffective in ferroptosis execution; consequent receptor silencing led to the consecution of erastin-induced ferroptotic cell death (Hasegawa et al., 2016), uncovering an additional pathway for ferroptosis modulation.

Overall, CD44v9-containing isoforms contribute to ferroptosis resistance through mechanisms involved in cysteine metabolism, which is necessary for ferroptosis initiation. Additionally, apoptosis regulation has also been observed. Further research on these pathways will facilitate the development of CD44v9-targeted therapies to overcome chemoresistance and improve cancer treatment outcomes.

Some approaches for CD44v9-exerted chemoresistance treatment entail the use of pharmacological inhibitors (Table 4). The sensitizing effect of SAS, a CD44v9-xCT signaling inhibitor, combined with cisplatin has been studied in pancreatic, hepatocellular and metastatic bladder cancer xenografts (Wada et al., 2018; Ogihara et al., 2019). It was demonstrated that combination treatment of xenografts with high expression of isoforms containing CD44v9 with SAS and cisplatin significantly decreased tumor growth and the number of tumor nodules compared to control cells, cisplatin alone and SAS alone. The current results indicate that such approaches may be useful for targeting CD44 variant isoforms to modulate chemoresistance and suggest alternatives/additives to standard chemotherapy regimens.

Notably, several cases have been reported surrounding the effect of CD44v9 expression on patient response to chemotherapy. For instance, in one study a patient treated with SAS for rheumatoid arthritis (RA), was further diagnosed with pT1 bladder cancer and underwent transurethral resection of bladder tumor (TURBT) (Takayama et al., 2016). They were further treated with MVAC and chemoradiotherapy due to the progression of the disease. With the appearance of a metastatic brain tumor, they underwent several courses of treatment with cisplatin and gemcitabine, and target lesions were classified as “complete response” shortly after the start of treatment. Interestingly, anti-CD44v9 staining of tumor tissue before TURBT and resected metastatic brain tumor unveiled a high expression and the absence of CD44v9 positive cells respectively, thus possibly attributing to the good clinical course outcome (Takayama et al., 2016).

In another study, a patient with prostate cancer underwent carboplatin-based chemotherapy (Takayama et al., 2016). During its administration, they were diagnosed with RA and PSA levels elevation was detected. Thus, alongside standard chemotherapy, SAS was administered, and surprisingly, PSA levels significantly decreased (by more than 50%) in just 2 weeks (Takayama et al., 2016). It should be noted that PSA levels further increased, although neuron-specific enolase (NSE) levels remained practically the same. Moreover, the prostate cancer tissue of the patient derived from needle biopsy and stained for CD44v9 demonstrated respective high expression levels, pointing to elevated resistance to chemotherapy (Takayama et al., 2016). Thus, CD44v9 expression levels may be of importance for favorable chemotherapy outcomes.

Moreover, clinical trials have been conducted regarding the targeting of CD44v9-containing isoforms (EPOC1205 and EPOC1407) (Figure 9). A CD44v9 pharmacological inhibitor SAS demonstrated a substantial decrease in CD44 variant isoform expressing cells by more than 10% in half of the patients and a reduction of intratumoral GSH levels in 70% of patients after its administration in combination with CDDP in advanced gastric cancer. However, only three out of eleven patients achieved the stabilization of disease, questioning the efficacy of such treatment. Another study demonstrated the insufficient efficacy of SAS in combination with cisplatin on advanced gastric patients clinical parameters–obtained objective response was not detected (EPOC1407) (Uppaluri et al., 2017; Shitara et al., 2017; Table 5). Moreover, one out of four patients achieved the stabilization of disease for more than 4 months. Thus, the effects of CD44v9 targeting by SAS are under question.