Cell Interactions and Signaling in Cancer

#### **Chapter 6**

### Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment

*Xing-Guo Li and Jer-Yen Yang*

#### **Abstract**

Hedgehog (Hh) signaling is a highly conserved pathway that plays a pivotal role during embryonic development. Mounting evidence has implicated Hh signaling in various types of cancer. Accordingly, inhibition of aberrant Hh signaling continues to be pursed across multiple cancer types -with some success in certain malignancies. In addition, with the renaissance of antitumor immunotherapy, an in-depth understanding of the molecular mechanisms underlying how the multifaceted functions of Hh signaling shape immunologically suppressive tumor microenvironment might be the key to unlocking a new era of oncological treatments associated with a reduced propensity for the development of drug resistance. Here, we focus on the latest advances regarding the immunological effects of misregulation of Hh signaling on tumor immunity. We also review the current status of clinically approved Hh inhibitors and dissect the mechanisms of drug resistance. Finally, we discuss the potential clinical applications that harness the immunomodulatory effects of Hh signaling not only to circumvent drug resistance, but also to achieve durable efficacy following immunotherapies, thus ultimately resulting in improved patient outcomes.

**Keywords:** hedgehog signaling, tumor microenvironment, immune cell, smoothened inhibitors, therapeutic targeting

#### **1. Introduction**

The Hedgehog (Hh) signaling pathway was discovered as a key regulator of organ development in *Drosophila melanogaster* by Christiane Nüsslein-Vollhard and Eric Wieschaus in the 1980s [1]. It was named after the gene locus associated with a spiky appearance of "hedgehog" phenotype in mutant *Drosophila* larve, findings based on which both investigators were awarded the Nobel Prize in Physiology or Medicine in 1995 "for their discoveries concerning the genetic control of early embryonic development," together with Edward B. Lewis [2]. Since then, the Hh signaling has been extensively studied as a highly conserved evolutionary pathway to orchestrate embryonic development, cell growth and differentiation, homeostasis [3]. Unlike other classical signaling cascades, Hh signaling is almost silent in the adult organisms but reactivated in a few tissues such as the skin, during tissue regeneration and wound healing [3]. Not surprisingly, aberrant activation of this pathway has been demonstrated as a potent oncogenic driver to promote numerous hallmarks of cancer [4]. Therefore, the multifaceted role of Hh signaling may allow exploitation of this key pathway for novel and more effective cancer therapy [5].

Activation of Hh signaling is dependent on the primary cilium, a highly specialized organelle found on most vertebrate cells. Three Hh ligands, sonic hedgehog (Shh), desert hedgehog (Dhh), and Indian hedgehog (Ihh), are known to actuate the Hh pathway during embryonic and tissue development [6]. Whereas the expression patterns for Dhh and Ihh are tissue-specific, Shh has a broader expression pattern in various compartments and in multiple developmental stages [6]. In general, the Hh signaling is activated through either canonical or non-canonical mechanisms. In the canonical pathway, Hh ligands bind to the surface receptor Patched 1 (PTCH1), which alleviates the inhibitory effect of PTCH1 on a G-protein-coupled receptor (GPCR)-like protein, Smoothened (SMO), leading to migration of SMO to the tip of the cilium, which in turn signals suppressor of fused (SUFU) to release glioma-associated oncogene homolog proteins (GLIs). Finally, GLIs translocate into the nucleus, resulting in a signaling cascade through transcriptional regulation of Hh target genes [6]. Alternatively, GLI transcription factors can be activated through non-canonical mechanisms, which can be independently of PTCH1, SMO, or both [6]. Of note, mounting evidence has demonstrated that the signaling pathways that can induce non-canonical Hh signaling have been of known significance in oncogenesis, providing the mechanistic basis of the cross talk between Hh signaling and other signaling pathways to promote tumorigenesis, as well as the rationale for development of potential combination therapeutics [7–10].

The discovery of PTCH mutations in basal cell nevus syndrome (BCNS, or Gorlin syndrome, or nevoid basal cell carcinoma [BCC] syndrome), a hereditary form of BCC, provides the first link between the Hh signaling and tumorigenesis [11, 12]. Other than BCC, emerging evidence has involved abnormal activation of Hh signaling in a variety of cancer types, such as medulloblastoma, breast cancer, pancreatic cancer, and lung cancer [13].

So far, three models have been proposed to elucidate the role of Hh signaling in oncogenesis where Hh signaling is over-activated through ligand production, autocrine, juxtacrine, or paracrine reception of the ligand, as well as cross talk between Hh signaling and complex intracellular signaling cascades [13]. First, in BCC and medulloblastoma, activating mutations of Hh pathway have been identified, such as inactivating mutations in PTCH or SUFU, and activating mutations in SMO, as shown in 85% of sporadic BCC or 30% of medulloblastoma, respectively [11–16]. In this scenario, the autonomous activation of Hh signaling is independent of Hh ligands.

Second, Hh signaling is aberrantly activated through autocrine or juxtacrine ligand-dependent manner, where Hh is secreted and responded by the same or adjacent cells [13]. This category of cancers includes breast cancer, pancreatic cancer, lung cancer, prostate cancer, colorectal cancer, stomach and esophageal cancer, ovarian and endometrial cancer, melanomas, and gliomas [13]. Finally, in pancreatic cancer, prostate cancer, and colon cancer, Hh signaling is activated through a paracrinedependent manner, where Hh ligands are secreted by tumor cells, whereas the PTCH receptor is expressed on stromal cells in the tumor microenvironment (TME). In this last model of Hh signaling activation, a feedback loop is generated, which allows the transmit of the growth-promoting signals from tumor cells to stromal cells and then back to tumor cells, leading to sustained tumor progression [17].

In the following sections, we will first highlight the key cellular components of TME involved in oncogenic Hh signaling to promote tumor progression. We will then review the current status of the FDA-approved and non-approved inhibitors of Hh signaling, as well as the molecular mechanisms of drug resistance. Finally, we will provide a critical evaluation of recent studies on the treatments combining immunotherapeutic strategies with approved Hh inhibitors and will propose potential strategies that could be applied to harness existing knowledge to overcome the drug resistance.

#### **2. The role of Hh signaling in the TME**

Emerging evidence has suggested that TME is not just a silent bystander, but rather an active player of tumor progression [18, 19]. The composition of TME not only varies between tumor types, but also is continuously evolving in the different stages of tumorigenesis. Hh signaling has been intensively studied with respect to the classical hallmarks of cancer [3–6]. In contrast, its role in the modulation of TME has only become evident in recent studies [20–22].

#### **2.1 Immune cells**

The adaptive and innate immune systems cooperate to form a highly proficient immune surveillance machinery that can identify and eradicate genetically altered cells to prevent tumorigenesis. Tumor-infiltrating leukocytes (TILs), including T and B lymphocytes, monocytes and macrophages, myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and natural killer (NK) cells, play diverse roles in tumor progression through interactions and production of cytokines, chemokines, and growth factors to support or suppress tumor growth and metastasis [20, 21]. There is increasing evidence from multiple experimental models that demonstrate an important and multifaceted role of Hh signaling in the modulation of immune cell functions. Aberrant Hh signaling induces a hostile, immunosuppressive microenvironment to dampen an effective antitumor immune response.

Regulatory T cells (Tregs) control the activity of effector immune cells such as granzyme B-expressing CD8+ T cells and NK cells by secreting anti-inflammatory cytokines such as TGF-β and interleukin-10 (IL-10) [23]. The immune modulatory role of Hh signaling in T cells is evidenced by recent studies demonstrating that Hh signaling may directly regulate the expression and activity of TGF-β. Treg infiltration has been described for Hh-associated tumors, such as BCC [24], and medulloblastoma [25–28].

Intriguingly, elevated Treg infiltration is accompanied by an increase of TGF-β within intra- and peri-tumoral skin in a human UV-exposed facial BCC model [29]. In line with the putative immunosuppressive phenotype of Hh signaling, genetic abrogation of T-cell TGF-β signaling mitigated tumor progression in a transgenic medulloblastoma mouse model overexpressing smoothened A1 (SmoA1), an obligatory and conserved Hh signal transducer [25]. In this study, TGF-β signaling blockade led to nearly abolishment of Tregs and licensing of CD8 cytotoxic T lymphocytes for antitumor immunity [23].

Mechanistically, GLI2, an Hh effector, has been shown to directly activate the expression of TGF-β in human Tregs [30]. Thus, it has been proposed that Hh signaling may help generate a feed-forward loop where TGF-β induces the inversion of CD4<sup>+</sup> T cells to Tregs, which in turn secrete high levels of TGF-β, leading to

enforcement of the continued presence of immunosuppressive Tregs in the tumor microenvironment [31].

Myeloid cell infiltration has been described in multiple cancer entities where tumors may benefit from myeloid cells-mediated immunosuppression. The role of Hh signaling in the tumor-promoting function of myeloid cells has been postulated based on observations in multiple models of Hh-induced tumors. First, in a murine SMOinduced BCC model, tumor growth appears to be enhanced by the recruitment of immunosuppressive myeloid derived suppressor cells (MDSCs), accompanied by a reduction of effector lymphocytes in the tumor lesions [32]. This is mediated by the TGF-β-CCL2 (C-C motif chemokine ligand 2) axis secreted by oncogenic SMOexpressing keratinocytes and the CCL2 receptor expressed by MDSCs. *In vivo*, pharmacological suppression of the CCL2 receptor expression decreased infiltration of MDSCs and resulted in reduced tumor growth, indicating an immunosuppressive phenotype by the oncogenic Hh signaling [33]. Likewise, there is also strong evidence for immunosuppressive function of myeloid cells in Hh-associated medulloblastomas, which are characterized by high myeloid infiltration. For example, gene expression profiling of human Hh medulloblastoma tumors showed enrichment for an M2-like gene expression profile, consistent with immunosuppressive functions of myeloid cells [34]. Moreover, an inverse correlation has been observed between expression of M2-like markers (such as CD163) and survival of human Hh medulloblastoma patients [34].

Along these lines, the notion of an immunosuppressive function of Hh signaling was further affirmed by two recent studies in Hh-induced medulloblastomas. In a mouse model of Hh medulloblastoma (*Ptch1*+/; *Tp53*/), Dang et al. showed decreased T-cell proliferation in a co-culture system of tumor-infiltrating myeloid cells and *ex vivo* stimulated T cells [35]. Mechanistically, the immunosuppressive phenotype appears to be mediated by CCL2. Genetic knockout of CCL2 receptor not only decreased infiltration of monocyte-derived macrophages but also increased levels of CD8<sup>+</sup> T cells in tumors [35]. Likewise, in another mouse model of Hh-induced medulloblastoma (Atoh1-SmoM2), pharmacological inhibition of colony stimulating factor 1 receptor (CSF1R) depleted macrophages and microglia, resulting in delayed tumor growth and prolonged mouse survival [36]. These recent studies support the notion of a tumor- promoting function of macrophages, which are consistent with an early study in another Hh-associated medulloblastoma tumor model, where the presence of MDSCs increases infiltration of Tregs and reduces the number of effector T cells [37]. Interestingly, infiltrating myeloid cells have been described as the predominant source of PD-L1 expression in a mouse model of Hh-induced medulloblastoma where the binding of PD- L1 to PD-1 on effector T cells resulted in T-cell exhaustion and immune escape of tumor cells [38]. Furthermore, an analysis of an immunocompetent breast cancer xenograft mouse model showed that inhibition of Hh signaling (SMO inhibitor vismodegib) led to reduced infiltration of immunosuppressive myeloid cells in the tumors, accompanied by an increase of effector CD8+ T cells and M1 macrophages [39].

#### **2.2 Tumor-associated astrocytes (TAAs)**

Astrocytes, the most abundant type of glial cells in the brain, are integral partners with neurons in the regulation of neuronal development and brain function. Hh signaling has emerged as a critical player to support astrocyte-mediated modulation of neuronal activity [40–42]. A recent series of elegant work supports a key role of

#### *Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108831*

tumor-associated astrocytes (TAAs) in promoting tumor growth and metastasis through distinct signaling, including Hh pathway [43–46]. First, TAAs were shown to secret the ligand Shh, which is required for maintaining cell proliferation of Hhactivated medulloblastoma through a Smo-dependent, but Gli1-independent manner, despite the absence of its primary receptor Ptch1. Of note, ablation of TAAs blocked tumor growth [43]. Furthermore, a recent study at single-cell resolution demonstrated that Hh-induced medulloblastoma cells can transdifferentiate into interleukin-4 (IL-4)-secreting TAAs, which in turn stimulates tumor-associated microglia to release insulin-like growth factor 1 (IGF1) to promote tumor progression [44]. Similarly, medulloblastoma-associated astrocytes have recently been shown to produce high levels of CCL2, a tumor-promoting cytokine shown to drive stemness maintenance and proliferation of disseminated tumor cells [45] and to promote metastasis [47]. Moreover, using single-cell RNA sequencing and lineage tracing analyses, Guo et al. investigated cellular origin of TAAs in a mouse model for relapsed Hh-activated medulloblastoma driven by *Ptch1* knockout [46]. This study has elegantly demonstrated that TAAs are predominantly derived from the transdifferentiation of tumor cells in relapsed MB, but not in primary MB, thus establishing the distinct cellular sources of astrocytes [46]. Interestingly, this study revealed that such transdifferentiation of medulloblastoma cells to TAAs depends on bone morphogenetic proteins (BMPs) and that pharmacological inhibition of BMP signaling repressed transdifferentiation and suppressed tumor relapse [46]. It remains to be determined what drives these transdifferentiation events and what intrinsic and extrinsic mechanisms, beyond Hh and BMP signaling, regulate the potential cooperation of TAAs and microglia in promoting the immunosuppressed state of medulloblastoma.

#### **2.3 Cancer-associated fibroblasts (CAFs)**

Cancer-associated fibroblasts (CAFs), the most abundant stromal cells in TME, have emerged as a central player in cancer progression and metastasis [48]. Through diverse phenotypes, origins, and functions, CAFs modulate the cross talk between inflammation and tumorigenesis and contribute to therapeutic resistance by producing various cytokines, chemokines, growth factors, and matrix-degrading enzymes [49].

There is increasing evidence indicating that CAF populations that support or suppress tumor growth and progression through stroma-specific Hh activation have been detected in multiple tumor types, including pancreatic cancer, colon cancer, and bladder cancer [50]. Recent advances in single-cell technologies have enabled detailed characterization of the heterogeneity and plasticity of differential CAF subsets, supporting a new therapeutic strategy in which tumor-supporting CAFs are reprogrammed into tumor-suppressing CAFs [50]. In pancreatic ductal adenocarcinoma (PDAC), Hh signaling pathway is activated in CAFs via a paracrine mechanism and has been associated with pancreatic tumorigenesis [49]. Initial studies indicated that inhibition of Hh pathway impaired tumor growth and sensitized tumors to chemotherapy in multiple PDAC models [51–56]. However, recent studies have challenged the concept of tumor-promoting CAFs. In the context of an oncogenic Krasdriven mouse PDAC model, conditional deletion of *Shh*, the predominant Hh ligand expressed in pancreas, led to cachexia and to poorly differentiated and highly vascularized tumors [57].

Moreover, by using three distinctly genetically engineered mouse PDAC models, another study showed that pharmacologic inhibition of Hh pathway activity

accelerated rather than delayed progression of oncogenic Kras-driven disease by affecting the balance between epithelial and stroma elements, leading to suppression of stromal desmoplasia but accelerated growth of pancreatic intraepithelial neoplasia [58]. These contradictory findings indicate that Hh signaling may play pleiotropic roles in PDAC progression. Interestingly, by using a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-offlight, and single-cell RNA sequencing, a more recent study defines dosage-dependent effects of Hh signaling on the composition and function of CAFs in PDAC microenvironment [59]. Hh signaling is uniquely activated and differentially elevated in CAFs, with higher levels in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF) in both mouse and human PDAC. Driving high levels of Hh signaling promotes tumor growth, whereas Hh pathway inhibition alters the ratio of myCAF/ iCAF populations, which is accompanied by a decrease in cytotoxic T cells and an expansion in regulatory T cells, thus altering the composition of CAFs, and shifting the inflammatory response toward a more immunosuppressive phenotype [59]. Given the differential functional implications for CAF subpopulations, changes in the ratio of CAF subtypes may lead to distinct antitumor outcomes. Consistent with, recent studies demonstrated a possible negative impact of current Hh pathway inhibitors on antitumor response in clinical trials, which were largely unsuccessful or even detrimental to patient health [60, 61]. Further understanding of the roles of Hh signaling in CAFs may open the possibility for more effective combination cancer therapies.

#### **3. Therapeutic targeting Hh signaling in cancers**

Given the multifaceted role of Hh signaling in cancer, inhibitors of Hh pathways have emerged as an important class of anticancer agents. These compounds fall into three main categories: Hh ligand inhibitors, SMO inhibitors, and GLI inhibitors [62]. Despite extensive efforts devoted to the discovery of Hh signaling inhibitors, so far only three drugs have been approved by the Food and Drug Administration (FDA), all targeting the upstream receptor of Hh signaling SMO, a membrane protein of the GPCR protein family [62].

#### **3.1 FDA-approved inhibitors**

To date, three SMO inhibitors, vismodegib, sonidegib, and glasdegib, have been FDA approved in 2012, 2015, and 2018, respectively, for cancer treatment. Cyclopamine, the first SMO antagonist, is a naturally occurring alkaloid found in the corn lily [63] later proved to bind to SMO and to inhibit activation of downstream Hh target genes [64].

Extensive efforts have been made to develop alkaloid derivatives to increase the bioavailability, sensitivity, and specificity of cyclopamine to target SMO [65]. Vismodegib (GDC-0449 or Erivedge), the first cyclopamine derivative and Hh pathway–targeting drug, is currently approved for the treatment of patients with locally advanced or metastatic BCC (US FDA). Compared to cyclopamine, vismodegib shows a higher potency and more favorable pharmacological properties [62]. The approval of vismodegib was based on results from the pivotal phase II ERIVANCE trial (Clinica lTrials.gov, NCT00833417) showing that vismodegib substantially shrank tumors or healed visible lesions (objective response rate, ORR) in 43% of patients with locally advanced BCC and 30% of patients with metastatic BCC, at 21 months, with a median progression-free survival (PFS) duration of 9.5 months for both metastatic and locally advanced BCC patients [66, 67]. Up to the completion of this manuscript, there have been 86 clinical trials for vismodegib, both monotherapy and combination, in various cancer types (ClinicalTrials.gov).

Sonidegib (Erismodegib, NVP-LDE-225, LDE-225, Odomzo) is another cyclopamine-derived SMO antagonist discovered in 2010 through an *in vitro*, highthroughput screen, showing high tissue penetration and bioavailability, as well as the ability to cross the blood-brain barrier [68]. In 2015, sonidegib became the second SMO inhibitor approved for patients with locally advanced or recurrent BCC (US FDA). The approval of sonidegib was based on results from a multicenter, randomized, double-blind phase II BOLT trial (ClinicalTrials.gov, number NCT01327053), which showed the objective response rates of 38% and 43% in the 800 and 200 mg dosage groups, respectively after 30 months in patients with locally advanced BCC and the objective response rates of 17% and 15%, respectively in those with metastatic BCC [69]. Up to August 2022, there are 46 clinical trials for sonidegib in cancer treatment (ClinicalTrials.gov).

A third FDA-approved inhibitor of Hh signaling is glasdegib (PF-04449913, Daurismo), a benzamide derivative discovered in 2012 with high potency and oral bioavailability [70]. In 2018, glasdegib was approved for combination treatment with low-dose cytarabine arabinoside (LDAC) for patients with acute myeloid leukemia unsuitable for intensive chemotherapy. The approval of glasdegib was based on the results of the phase II BRIGHT 1003 trial (ClinicalTrials.gov, NCT01546038) showing the median overall survival of 8.8 months with glasdegib/LDAC as compared to 4.9 months with LDAC. Furthermore, 17.0% and 2.3% of patients in the glasdegib/ LDAC and LDAC arms, respectively, achieved complete remission [71]. Up to this point, there have been 26 clinical trials for glasdegib in various cancer types (Clinica lTrials.gov).

#### **3.2 Resistance mechanisms to FDA-approved inhibitors**

The first retrospective study on drug resistance to SMO inhibitor therapy was reported in 2012 where 21% of BCC patients treated with vismodegib developed drug resistance, with a mean tumor recurrence time of 56.4 weeks in clinical examination [72]. Ever since, resistance to SMO antagonists has been observed in patients who never respond to SMO inhibitor therapy (primary resistance), as well as in those who initially respond but later develop resistance to SMO inhibitors (acquired resistance) [73]. Mechanistically, a number of models have been proposed to explain the basis of drug resistance to SMO inhibitor therapy. First, genetic analysis of resistant tumors has revealed mutations of SMO, loss of SUFU, and amplification of GLIs or Hh target genes, such as CCND1 and GLI1 [5, 10]. Second, accumulating evidence supports the notion that the resistance can be driven through the non-canonical Hh signaling, accompanied by the concurrent activation of other oncogenic signaling pathways, such as AP-1 and TGF-β signaling [74], RhoA signaling [75], and RAS-MAPK signaling [76]. Finally, a new mechanism has recently been uncovered to contribute to drug resistance through loss of primary cilia [77, 78]. This was supported by both preclinical and clinical evidence. In Hh-dependent medulloblastoma, recurrent mutations in oral facial digital syndrome 1 (OFD1), a culprit gene led to loss of cilia, and thereby caused resistance to SMO inhibitors [78]. Importantly, sequencing data analysis from resistant BCC patients showed recurrent mutations in ciliary genes, providing clinical relevance of this new mechanism [77]. Therefore, a better understanding of ciliaregulating signaling pathways in resistant cancer may open up a new route to reintroduce cilia to sensitize resistant cancer cells to SMO inhibitors. Taken together, several strategies have been proposed to overcome the drug resistance through targeting the underlying mechanisms. These approaches include: (1) develop secondgeneration SMO inhibitors to retain anticancer activities that are not affected by the resistance-inducing mutations [5]; (2) target downstream components of SMO, such as GLIs (see below, non- approved inhibitors), or signaling molecules involved in the non-canonical Hh signaling pathway [8].

#### **3.3 Non-FDA-approved inhibitors**

Multiple novel inhibitors targeting SMO have been shown to be effective in preclinical models [5] and are now in active clinical trials, either monotherapy or combination for various cancer types. These compounds include saridegib (patidegib, IPI-926), taladegib (LY2940680), and BMS-833923 (XL139) (ClinicalTrials.gov). On the other hand, even though GLI1 antagonists are not as extensive as those targeting SMO, mounting evidence has shown that targeting the Hh signaling at the level of its final effector, GLI1, is a promising strategy to overcome resistance to currently available SMO inhibitors [79, 80]. In this regard, the promising pharmacological potential of direct and indirect GLI inhibitors, as well as GLI antagonists derived from natural products, has been in active investigation at the preclinical or clinical phase. It is anticipated that future study on these compounds will help develop new strategies tackling resistant mechanisms and tumor heterogeneity [81].

#### **4. Hh signaling and antitumor immune response**

In 2018, James P Allison and Tasuku Honjo were awarded the Nobel Prize in Physiology or Medicine "for their discovery of cancer therapy by inhibition of negative immune regulation" [82]. Although this breakthrough in cancer immunotherapy has revolutionized cancer treatment, only a subset of patients elicit favorable responses and most immunologically cold solid tumors are not responsive [83]. Given the immunosuppressive function of Hh signaling, inhibitors of Hh signaling pathway may hold promise in converting nonresponsive cold tumors into responsive hot ones, which may subsequently allow nonresponders to benefit from immunotherapies. Notably, clinically approved Hh inhibitors, as well as non-approved inhibitors, have been in active preclinical and clinical trials for combined therapies, including immunotherapies.

The first clinical trial with Hh inhibitors in combination with immune checkpoint inhibitors was conducted in 16 patients with advanced BCC (clincialtrial.gov, NCT02690948). This trial showed that pembrolizumab (PD-L1 inhibitor) is active against BCCs. Although the two groups of pembrolizumab with or without vismodegib were not directly compared, the response rate for the combination group was not superior to the monotherapy group [84]. Of note, most patients with advanced BCC progress on or are intolerant to Hh inhibitor therapy despite objective response rates of 30–60% [66–69, 85]. Until Feb 9, 2021, when cemiplimab, a PD-1 antibody, was approved by the US FDA fully for patients with locally advanced BCC, and accelerated for patients with metastatic BCC, after treatment with Hh inhibitors, or for whom Hh inhibitors are not appropriate [86], there was no standard second-line treatment option for these BCC patients [72]. A recent clinical trial study provides the first report to show clinically meaningful antitumor activity of cemiplimab in patients with BCC after Hh inhibitor therapy ([87], clinicaltrials.gov, NTC03132636). In this trial, the efficacy and safety of cemiplimab were evaluated in patients with locally advanced BCC or metastatic BCC who had previously been treated with an Hh inhibitor. Among the efficacy population (n = 121), centrally reviewed objective response was observed in 31% of patients with estimated duration of response exceeding 1 year in 85% of responders [87].

Importantly, this study also showed that the safety profile was consistent with what is known for immune checkpoint class of drugs, even considering the advanced age of the patient population in the present study [87]. These findings demonstrate the efficacy of immune checkpoint blockade in treating BCC in patients who had previously received Hh inhibitor therapy, thus opening a new horizon for treatment of the many patients who discontinue Hh inhibitor therapy due to disease progression, toxicity, or drug resistance. Moreover, a recent case report demonstrated an impressive response to cemiplimab in a sonidegib-resistant giant basosquamous carcinoma, one form of BCC [88]. Finally, a dozen of clinical trials have been initiated to investigate the combination treatment of anti-PD-1, PD-L1, and CTLA-4 monoclonal antibody therapy with first-line Hh inhibitors in patients with a variety of cancer types (see **Table 1**). The outcome of these trials will not only inform about whether combinatorial treatments can increase the efficacy and duration of antitumor response, but also provide insights into the optimal customized regimen to circumvent resistance to Hh inhibitors.

Comparatively a few recent studies have indicated possible negative effects of the current Hh inhibitor therapy on antitumor immunity [89]. For instance, blockade of SMO signaling may inhibit formation of the immunological synapse [90]. Administration of SMO inhibitors caused the functional disruption of the immunological synapse, leading to the loss of T-cell effector activity [90]. Even though it remains unclear whether Hh inhibitor therapy may impede cytotoxic T-cell killing in cancer patients, a pilot clinical trial study of vismodegib in combination with pembrolizumab did not suggest additive clinical activity [84]. In the clinical context, there is an emerging paradigm that immunotherapy may show the greatest activity when administered early in the natural history of cancers. Further studies are warranted to evaluate the efficacy and duration of immune checkpoint blockade before Hh inhibitor therapy.

#### **5. Conclusions**

The Hh signaling pathway has attracted extensive research attention as a key player to contribute to the progression of a variety of human cancer types. With an indepth understanding of the molecular mechanisms underlying the role of Hh signaling in tumorigenesis, enormous efforts have been made to develop specific inhibitors targeting molecular components of this pathway. Consequently, cancer therapy has undergone a paradigm shift from eradicating tumor cells to multidimensional targeting and normalizing tumor cells and TME. Herein, we reviewed the multifaceted function of Hh signaling in shaping immunologically suppressive TME to promote tumor progression, provided an up-to-date status of active clinical trials of FDA approved Hh inhibitors, and finally, highlighted possible therapeutic interventions that harness the immunomodulatory effects of Hh signaling not only to overcome drug resistance, but also to achieve durable efficacy following immunotherapies.


#### *Tumor Microenvironment – New Insights*


#### *Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108831*

**SMO** 


*Tumor Microenvironment – New Insights*


#### *Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108831*


#### **Table 1.**

*Combination therapy of SMO inhibitors under clinical trials.*

#### **Acknowledgements**

This book chapter was in part of supported by MOST 110-2311-B-039-001, MOST 111-2320-B-039-050 and CMU109-YT-03 (X.-G.L.) and MOST 111-2320-B-039-015 and CMU109-YT-02 (J.-Y.Y.).

### **Author details**

Xing-Guo Li1,2 and Jer-Yen Yang1,2\*

1 Graduate Institute of Biomedical Science, China Medical University, Taichung, Taiwan

2 Institute of Biochemistry and Molecular Biology, China Medical University, Taichung, Taiwan

\*Address all correspondence to: jyyang@cmu.edu.tw

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108831*

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*Perspective Chapter: Critical Role of Hedgehog in Tumor Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108831*

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#### **Chapter 7**

## Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis

*Anyu Gu, Chikezie O. Madu and Yi Lu*

#### **Abstract**

The tumor microenvironment consists of multiple types of cells, including endothelial cells, pericytes, neutrophil macrophage mast cells, lymphatic cells, basement membrane extracellular matrix, as well as fibroblasts. Fibroblasts populations found in cancers, also known as cancer-associated fibroblasts, have been implicated in the initiation, progression, and metastasis of tumors. This chapter will focus on the roles of cancer-associated fibroblasts in the progression of cancer and the studies of use of cancer-associated fibroblasts as a therapeutic target for cancer intervention.

**Keywords:** tumor microenvironment, cancer-associated fibroblasts, fibroblasts, cancer intervention, cancer

#### **1. Introduction**

The tumor microenvironment (TME) is the environment in which tumor cells or cancer stem cells exist [1]. The TME consists of multiple types of cells, including endothelial cells, immune cells, and fibroblasts [1–3]. The TME also consists of components such as the extracellular matrix (ECM), soluble factors such as cytokines and growth factors, and physical properties such as pH and oxygen content [2]. The TME and the interactions between its components help to promote tumor growth and cancer progression (**Figure 1**) [3].

Fibroblasts are the most common type of cell in connective tissue, commonly defined as structural cells that specialize in depositing and remodeling the ECM [4]. Fibroblast populations found in primary and metastatic cancers, known as cancer-associated fibroblasts (CAFs), are implicated in tumor initiation, progression, and metastasis [5]. CAFs have wide varieties of cells-of-origin, heterogeneous phenotypes, and diverse functions, all of which are shared by other cells found in the TME [6]. This chapter will focus on CAFs and their potential use in cancer intervention.

**Figure 1.** *Important interactions and mechanisms of the TME [1].*

#### **2. Fibroblasts and cancer-associated fibroblasts**

The precursors of CAFs are generally considered to be dormant tissue-resident fibroblasts and pancreatic and hepatic stellate cells, though different studies have also identified bone marrow-derived mesenchymal stem cells, endothelial cells, and adipocytes [5]. Fibroblasts play a prominent role in coordinating the wound repair response in skin; therefore, it is likely that key CAF traits correspond to the normal physiological role normal fibroblasts play [7]. Fibroblasts transform into CAFs through tumor-derived stimuli, including soluble factors secreted by the tumor, immune infiltrate, lysophosphatidic acid, fibroblast growth factor, interleukin-1 (IL-1), IL-6, and granulin [8]. Transforming growth factor β (TGFβ) and lysophosphatidic acid are well-established activating signals for fibroblasts, which promote the activity of SMAD transcription factors and serum response factors, respectively [7]. These fibroblast activating signals converge to drive expression of the fibroblast marker αSMA, as well as increase the activity of the contractile cytoskeleton [7]. Fibroblasts may become activated through Notch signaling when in direct contact with tumor cells [7, 8]. Other mechanisms that can activate normal fibroblasts to become CAFs are shown in **Figure 2**. In the TME, tumor cells secrete factors such as TGFβ, platelet-derived growth factor (PGDF), and fibroblast growth factor (FGF) to convert fibroblasts to CAFs [3]. A build-up of CAFs is often associated with poor prognosis in many cancer types [3].

As shown in **Figure 3** [10], TGFβ is a common factor in the conversion of many different cell types into fibroblasts and CAFs. There are many types of TGFβ. TGFβ-1 is one that is secreted by stromal and tumor cells and is the main factor in promoting the mobilization of residential fibroblasts and their activation into CAFs [10]. Through SMAD-dependent and SMAD-independent pathways, TGFβ-1 activates fibroblasts into CAFs, expressing alpha-smooth muscle actin, periostin, α-fibroblast

*Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis DOI: http://dx.doi.org/10.5772/intechopen.108832*

#### **Figure 2.**

*Mechanisms that activate normal fibroblasts to become CAFs. FGF, fibroblast growth factor; PDGF, plateletderived growth factor; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; TGFβ, transforming growth factor-β; TNF, tumor necrosis factor [7].*

#### **Figure 3.**

*Different origins of CAFs [9, 10].*

activation protein, and fibroblast-specific protein-1 [10]. In addition, activated fibroblasts secrete TGFβ-1 [10], which could create a positive feedback loop, increasing fibroblast activation. TGFβ binds to the type 2 of TGFβ receptor (TGFBR2) on the surface of fibroblasts [11].

#### **2.1 CAFs in tumors**

CAFs are a type of myofibroblast that enhance the malignancy and progression of cancer [12]. The presence of CAFs is identified in almost all solid tumors [13]. This suggests that CAFs are important to the formation of solid tumors. In an established tumor, the TME represents a changed part of the original normal tissue of the host [13]. Tumor cells mostly contribute to the change in their favor. [13]. The stromal transformation of TME is primarily dominated and maintained by CAFs [13]. The CAF component of the TME is the most critical in influencing most of the functions of the TME in real time [13]. CAFs alter the TME by directly interacting with cancer cells and regulatory paracrine signaling, control the immune response to neoplasia, deposit ECM components, stimulate angiogenesis, and provide a scaffold for tumor invasion and metastasis [14]. Additionally, CAFs can produce many growth factors and pro-inflammatory cytokines to promote angiogenesis and recruit immunosuppressive cells to the TME to evade the immune system [9].

While CAFs have historically been considered to be cancer-promoting components, recent studies have shown that CAFs could have tumor-restraining functions in certain circumstances [15]. The tumor-restraining actions of CAFs are likely dependent on stimulation of anticancer immunity, pro-inflammatory secretome, tumor inhibitory signaling, and the synthesis of ECM components as barriers to tumor cell invasion and dissemination [5]. A study in mice has shown that myofibroblast depletion leads to increased tumor invasion, which is associated with decreased survival [16]. This study suggests that CAFs have functions in restraining tumors. This paradoxical nature of CAFs can potentially be explained by the heterogeneity of CAFs [15].

#### **2.2 Heterogeneity of CAFs**

There is mounting evidence that CAFs are a heterogeneous population of cells [9]. This likely depends on the numerous precursors of CAFs [9]. CAFs can be recruited to the tumor from a distant source, such as bone marrow [14], or transdifferentiate from non-fibroblastic lineages, such as epithelial cells, blood vessels, adipocytes, pericytes, and smooth muscle cells [9]. Numerous precursors of CAFs are shown in **Figure 3**. The study of genetically modified mouse models (GEMMs) designed to limit the accumulation of CAFs in growing pancreatic tumors or to conditionally delete the vascular endothelial growth factors in breast CAFs revealed that there are distinct functional subtypes of CAFs [17].

#### **2.3 Functions of CAFs**

CAFs have both pro-tumor and antitumor tendencies [17]. Pro-tumorigenic functions of CAFs are generally driven by their altered secretive [17]. Paracrine signaling between cancer cells and CAFs leads to tumor progression by enhancing the survival, proliferation, stemness, and metastasis-initiating capacity of cancer cells, promoting cancer progression and enhancing resistance to therapy [17]. CAFs also have an indirect influence in promoting tumor growth due to their ability to remodel the ECM [17]. The stiffness of the tissue, which plays a critical role in tumorigenesis, is influenced by modifications in the ECM's composition and cross-linking [18, 19]. CAFs express lysyl oxidase (LOX), an enzyme that cross-links and stiffens collagen fibers, promoting their stability [18]. CAFs also regulate the degradation of the ECM

#### *Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis DOI: http://dx.doi.org/10.5772/intechopen.108832*

[18]. CAFs secrete cytokines and chemokines that regulate tumor immunity and the intratumoral vascular program [17]. Several studies have indicated that CAFs play an important role in chemoresistance *via* different mechanisms, including but not limited to increasing stem cancer cells, secreting cytokines, and secreting miRNAs [10]. miRNAs have been shown to inhibit tumor-repressor genes, thus promoting cell growth and invasion, metastasis, and tumorigenesis [20].

CAFs trigger tumor initiation and progression [18]. *In vitro* coculture and *in vivo* transplantation experiments have shown that human prostatic CAFs induced the proliferation and the ability to form tumors from immortalized nontumorigenic human prostatic epithelial cells [18]. This effect was not exhibited by normal fibroblasts. It is thought that CAFs' secreted factors are what cause this tumor-initiating potential [18].

Numerous studies have shown that CAFs confer resistance to chemotherapy [6]. Some CAF-mediated resistant mechanisms include delivery of exosomes stimulating cancer cell survival, promoting cancer cell epithelial-mesenchymal transition, and thus decreasing expression of transporters responsible for drug uptake and scavenging chemo drug to reduce the amount of intratumoral chemotherapy drug [6]. CAFs also contribute to the resistance to targeted therapy [6]. Additionally, evidence suggests that CAFs contribute to immune evasion and immunotherapy resistance [6].

Antitumor functions of CAFs are predominantly associated with their functions as regulators of antitumor immunity [17]. Studies in mice have shown that fibroblast depletion leads to increased tumor invasion [16]. The use of defined gene promoterdriven expression of viral thymidine kinase proteins in GEMMs to study CAFs has allowed researchers to deplete populations of CAFs using ganciclovir, a substance that is toxic only to cells that express viral thymidine kinase [17]. A similar approach to deplete CAFs expressing αSMA suggested that αSMA+ stromal cells were predominantly acting to restrain cancer progression [17]. The depletion of these αSMA expressing CAFs yielded a more invasive tumor with enhanced intratumoral hypoxia [17]. A reduction in CAFs in GEMMs of pancreatic tumors with a deletion of sonic hedgehog (SHH) in the cancer cells also resulted in more aggressive tumors with increased cancer proliferation [17].

#### **3. Targeting CAFs for cancer intervention**

Numerous studies have proven CAFs' significant role in cancer progression and subsequently the potential of CAFs as targets for effective cancer intervention. Traditionally, therapies involved targeting cancer cells directly [21]. Recent complementary efforts aim to disrupt the networks that promote cancer cell activity and behavior [21]. The depletion of CAFs and targeting of CAF-dependent pathways can indirectly result in malignant cell death through both immune-dependent and immune-independent mechanisms [21]. Most conventional cancer therapies, such as radiotherapy and chemotherapy, are likely to affect CAFs as well by preventing cellular division by inducing DNA damage, impeding DNA and RNA synthesis, and blocking the cytoskeleton remodel required for cell division [17]. However, the unintended impact of these therapeutic methods on the function and accumulation of CAFs is largely unknown [17].

As a result, research is being conducted to help target CAFs through alternative methods [21]. One approach involves targeting the regulatory pathways leading to fibroblast differentiation and activation [9, 17, 21]. For example, TGFβ is a common


**Table 1.**

*Summary of various drugs' efficacy against CAF-induced cancer progression in clinical and pre-clinical studies [6].*

factor in the conversion of different cell types into CAFs. In a study, Mariathasan et al. found the two top scoring TGFβ pathway genes represent a ligand, TGFβ1, and receptor TGFβR2 [22]. In murine tumor models, blocking the TGFβ signaling by using the SM16 TGFβ receptor inhibitor or anti-TGFβ antibodies resulted in the recession of tumor growth [23]. By targeting these regulatory pathways, the activation of fibroblasts could be prevented, preventing CAFs from activating and functioning.

*Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis DOI: http://dx.doi.org/10.5772/intechopen.108832*

Another approach for targeting CAFs for cancer intervention is targeting CAF-secreted factors [11]. Numerous mitogens, chemokines, and matricellular proteins that CAFs release aid in the evolution of tumor progression and the development of drug resistance [11]. Targeting these CAF-secreted factors should prevent the promotion of tumor progression and drug resistance, making the tumor more susceptible to drugs. The heterogeneity of CAFs also proves as an advantage for cancer intervention *via* shifting the influence of pro- vs. antitumorigenic populations [21].

Currently, there are many drugs under trial as shown in **Table 1**. Of the potential targets identified in CAFs, fibroblast-activation protein (FAP) is the most studied. FAP has been neither detected in benign tumors nor in most normal quiescent adult stromal cells [35]. FAP is a type II integral membrane of the prolyl oligopeptidase family, or S9 family [36]. FAP is further classified into the dipeptidyl peptidase (DPP) subfamily (S9B) [36]. This class of enzymes is characterized by its capacity to cleave the pro-Xaa peptide link, where Xaa can be any amino acid. It has been demonstrated that this enzymatic activity contributes to the development of cancer by altering bioactive signaling peptides [36]. *In vivo,* FAP+ CAFs were successfully depleted by the FAP-depleting immunotoxin, and tumor models demonstrated strong tumor inhibitory effects. [6]. Other approaches in targeting FAP include DNA vaccine and chimeric antigen receptor (CAR) T cells [6].

#### **4. Conclusions**

Heterogeneous populations of CAFs exist in the TME. The heterogeneous nature of CAFs likely comes from their different origins, and this heterogeneity is likely the cause of the paradoxical nature of CAFs having both pro-tumorigenic and antitumorigenic functions. CAFs have many functions in the TME. CAFs alter the TME. They produce growth factors and pro-inflammatory cytokines.

CAFs are a promising target for cancer intervention. They have many pro-tumorigenic functions. CAFs can be targeted through their activation pathway by blocking a step in the pathway. One method is by preventing FAP from being produced by introducing siRNAs that are complementary to the FAP mRNA.

Over time, our understanding of CAFs and their contribution to cancer progression has expanded greatly. We now have a better understanding of their heterogeneity and their functions in the TME. While the antitumorigenic functions may act as a roadblock to targeting CAFs for cancer intervention, it may be possible to develop a treatment that targets the pro-tumorigenic functions of CAFs without targeting the antitumorigenic functions of CAFs by targeting subpopulations of CAFs that express pro-tumorigenic genes.

While CAFs are a promising target for cancer intervention due to their pro-tumorigenic functions, their antitumorigenic functions may act as a roadblock. Further research would be required before targeting CAFs as a conventional method of cancer intervention.

However, there are issues with targeting CAFs for cancer intervention. **Figure 4** shows the effects of targeting only tumor cells vs. targeting only the TME on a tumor. In both, there is a possibility that the tumor can grow back. In addition, studies in mice have shown that fibroblast depletion leads to increased tumor invasion [16]. In mouse models, deletion of SHH accelerated the progression of pancreatic ductal adenocarcinoma [37].

#### **Figure 4.**

*A schematic diagram of targeting tumor cell or TME only and their potential resistance mechanisms. Left: Targeting tumor cells only (such as chemotherapies) kills majority tumor cells. However, the residue tumor cells may survive due to the TME, leading to tumor relapse. Right: Targeting the TME can inhibit the recruitment and activation of pro-tumor cells and enhance antitumor responses. However, the TME will be reconstituted by tumor cells via recruitment and programming of bone marrow derived cells or local resident stromal or immune cells [6].*

#### **Acknowledgements**

Funding for the publication of this paper was made possible by a grant from The Assisi Foundation of Memphis. Brown, Chester, PhD (PI). We also thank Yirui Tang for drawing the figures used in this paper.

#### **Conflict of interest**

The authors have declared that no conflict of interest exists.

*Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis DOI: http://dx.doi.org/10.5772/intechopen.108832*

### **Author details**

Anyu Gu1 , Chikezie O. Madu<sup>2</sup> and Yi Lu3 \*

1 Departments of Biology, White Station High, Memphis, TN, USA

2 Departments of Biological Sciences, University of Memphis, Memphis, TN, USA

3 Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA

\*Address all correspondence to: ylu@utshc.edu

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Ahmed Lasfar*

This book offers outstanding approaches to understanding the role of the tumor microenvironment (TME) in cancer development and metastasis. The TME, with its multifaced role, is fundamental in the control and exacerbation of almost all cancer types. The outcome of many solid tumors is dependent on the modulation of the TME. Local tumor immunity, which is crucial in the control of cancer promotion, is one of the leading compounds of the TME. This book presents new insights and provides detailed and updated descriptions of the role of the TME in the control and the development of almost all cancer types. This book is an authentic source of knowledge, useful for researchers, medical doctors, students, and all individuals interested in understanding the mechanisms of cancer control and development.

Published in London, UK © 2023 IntechOpen © Marcin Klapczynski / iStock

Tumor Microenvironment - New Insights

Tumor Microenvironment

New Insights

*Edited by Ahmed Lasfar*