Lung Cancer is one of the leading causes of death across the world [1]. Based on histology, lung cancer can be classified into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC accounts for around 14% of all lung cancers [2] and each year, around 30,000 people are diagnosed with SCLC [3]. Tobacco exposure remains the primary risk factor and this risk increases with the intensity and duration of tobacco usage [4]. This cancer originates from the pulmonary neuroendocrine cells (PNECs) or Kulchitsky cells (K cells) and is associated with mutation-induced loss of function of the tumor suppressor genes like Rb and Trp53 [5]. SCLC shows early metastasis to various organs like adrenal glands, brain, bone, lymph nodes, and liver. Both the rapid growth and high metastatic nature results in a high mortality rate in patients suffering from SCLC [6]. The survival chances barely increase after undergoing treatment. After initial treatment, less than 5% of the patients could survive for more than 5 years and this period is even brief for treatment-naive patients with an average overall survival period of only 2–4 months [7]. Also, the standard treatment strategy for SCLC has not been changed in the last few decades [8].

Based on the extent to which cancer had spread from its primary site, SCLC can be staged as an Extensive stage disease (ED) and Limited stage disease (LD). Generally, we call it LD when the tumor is confined to one lung and if it spreads beyond that, it is said to be ED. Generally, both the LD and ED patients give a satisfactory response to initial chemotherapy and radiotherapy but most of them show a relapse in 6–12 months [9]. The LD-SCLC patients are normally treated with 4–6 cycles of chemotherapy with carboplatin or cisplatin and etoposide along with thoracic radiotherapy [10]. For the ED-SCLC patients, combined chemotherapy of carboplatin plus etoposide is administered that yields a satisfactory response in 60–70% of patients. Nearly 10% of these patients showed a complete radiographic response to initial chemotherapy and are subsequently considered for PCI and/or thoracic radiotherapy [10]. Importantly, prophylactic cranial irradiation (PCI) and chest irradiation (CRT) were successful in increasing the survival rate [6, 11]. Prophylactic Cranial Irradiation (PCI) is generally recommended for those who demonstrate a satisfactory response to the initial therapy. It was noted that 90% of the patients respond to such treatment while 25% of them gain long-term survival [10]. Since the development of active chemotherapy regimens 4 decades ago, the overall survival of SCLC patients had slightly improved. The 2-year survival rate of LD-SCLC patients has been improved from 15 to 22% with a slight increase (3.4 to 5.6%) for ED-SCLC patients during the period of 1973–2002 [10]. Over the last 30 years, a number of treatment protocols have failed to treat ED-SCLC. After the approval for Topotecan as second-line treatment, it took more than 20 years to get FDA approval for Nivolumab in 2018 [12].

Various developmental signalling pathways like Notch, hedgehog play an important role in cellular functions including cell motility, lineage, apoptosis, etc. It has been observed that these pathways also drive the initiation and maintenance of SCLC cells [13]. These pathways are very crucial in regulating self-healing in case of injury [14]. Thus, giving an indication that misregulation of these pathways may contribute to tumor growth. These characteristics have paved a path to explore these pathways as potential drug targets for cancer treatment. In this article, we have highlighted several challenges that are faced by the research fraternity to propose a new treatment for SCLC. Also, it provides a first comprehensive review on the roles of various developmental pathways for the initiation and growth of SCLC along with the attempts made by various research groups to reduce the progression of this cancer by targeting these pathways.

For the last 15 years, there has been no improvement in survival rates and no new treatment has been approved. Here, we have highlighted some major issues that make the treatment difficult and challenging for SCLC.

No early diagnostic method: Due to the rapid growth and early metastatic spread, most of the SCLC patients are diagnosed at a later stage when the disease gets widespread. As per the report of the National Lung Screening Trial conducted in 2002, early-stage SCLC could not be detected by CT scan. It is very unfortunate that no effective diagnostic method is available to date for the early detection of SCLC [15].

Non-availability of sufficient tumor tissue for diagnosis and study of SCLC: Only a very small amount of tumor tissues like fine-needle aspirates, core biopsies, and bronchoscopic biopsies are available to perform the translational research to develop the diagnosis and treatment process of SCLC. Also, the small cell size makes the handling of these cells difficult as they can be easily distorted using forceps or aspiration needles used during sample collection [16]. Though the majority of the SCLC patients are treated by chemotherapy and radiotherapy, a small number of patients undergo surgery to remove the tumor. Since it requires robust materials for analyzing molecular profiling like genome or exome sequencing, the non-availability of tissues limits such study for SCLC. Therefore, it was not possible to include SCLC in The Cancer Genome Atlas (TCGA) [15].

The paucity of animal models: The non-availability of animal models also limits the study on SCLC. There are only a few genetically engineered mouse models available. However, none of these models can fully mimic the genetic complexity of SCLC in humans. It is basically due to the lower mutational burden for the non-exposure of tobacco to the animal model. These models are very costly and demand a great deal of time for the development of cancer. It requires more than 9 months for developing models with inactivated Trp53 and Rb1 [17]. In addition, it was noticed that during the transfer of cancer cells from one xenograft model to another, certain mutations that were present in the parent cell line were found missing in the transferred model [15].

Complex molecular pathogenesis of SCLC: Due to high exposure to tobacco carcinogens, the molecular pathology of SCLC becomes highly complex with lots of genetic alterations like insertions, deletions, mutations, gross inter-and intra- chromosomal rearrangements, and large-scale copy number alterations. Most of the mutations obtained in SCLC are passenger mutations indicating that these don’t significantly contribute to the growth and progression of the disease. Since only a few mutations are identified in all the cases, it gives very few therapeutic targets for the treatment. It was noticed that the mutations of TP53 and RB1were found in 75–90% and 60–90%, cases respectively. The underlying mechanistic details that contribute to the elevated metastatic behaviour of SCLC are still unknown [15].

Resistance to chemotherapy: The majority of LD-SCLC and ED-SCLC patients experience a relapse in just 6–12 months after completion of initial treatment. Both the molecular mechanisms involved in the development of resistance and the chemoresistant properties of the cell population are yet to be discovered [15].

Lack of SCLC-specific research projects: The research work with SCLC has been dramatically decreased for the lack of tumor tissue and animal models. It was clearly reflected on the National Cancer Institute (NCI) research portfolio that considers only 17 projects (approx. 2%) for SCLC out of 745 projects on lung cancer research [15].

Developmental signalling pathways are crucial for proper embryonic development, are highly conserved and expressed in most multicellular organisms [18]. The deregulation of these pathways may lead to tumor initiation and progression among adults [18, 19]. In the case of various tumors, the misregulation of these pathways may also lead to metastasis, drug resistance, and angiogenesis [19]. Accordingly, these pathways are gaining importance as potential drug targets especially in the case of advanced and metastatic tumors. SCLC is a rapidly growing tumor with a very quick stage transition time. Furthermore, in most cases, this type of cancer is identified in the patients when it has already reached its extensive stage (ED). In addition, the tumor cells become resistant to chemotherapy even after the initial treatment [20]. Vowing to all these factors, here we have tried to provide an understanding of the roles played by the developmental signalling pathways for initiation and progression of SCLC. We have also briefly mentioned the efforts that have been made in this respect to counter this deadly tumor.

4.1 Hedgehog signalling pathway

Hedgehog (Hh) signalling pathway has a central role in controlling the cell fate, tissue polarity, and organogenesis during development [21]. The Hh pathway is highly regulated in adult tissues under normal conditions. It can also play a vital role in early lung development by epithelial-mesenchymal interactions [21, 22]. However, in the case of cancer, it is observed that there is a misregulation in this pathway. The canonical pathway can be activated by Hh glycoproteins like Sonic (Shh), Indian (Ihh), and Desert (Dhh) that act as ligands for the Hh pathway. Among these, Shh is the most potent ligand [23]. When the Hh ligands are absent, the Patched1 (PTCH1), a 12-pass transmembrane receptor inhibits the progression of the pathway. Further, the binding of Shh to PTCH1 relieves the repression of Smoothened (SMO) protein that helps in its translocation into the primary cilium. This helps in the activation of glioma-associated transcription factors (GLI1, GLI2, GLI3) via initiation of the signalling cascade. When SMO is inactive, GLI is bound to Suppressor of fused (SUFU) due to which GLI remains in an inactive state. As a result, this complex gets accumulated in the cytoplasm which ultimately results in its degradation [24]. In the active state, the GLI dissociates from the SUFU and is transported to the nucleus where it regulates the transcription of the associated genes (Fig. 1A) [25].

Fig. 1.

Role of Hedgehog, Notch and FGF signalling pathways in SCLC and their therapeutic interventions. (A) Hedgehog pathway – The pathway is initiated when the ligand shh binds to the patched1 receptor (PTCH1). This relieves the inhibition on the Smoothened (SMO) protein that is responsible for inhibiting Suppressor of Fused (SUFU). SUFU prevents the entry of Glioma associated Transcription Factor (GLI) into the nucleus when activated. Thus, SUFU inhibition releases the bound GLI into the cytoplasm after which it is transported to the nucleus and acts as a transcription factor. The therapeutics Vismodegib, Erismodegib, Cyclopamine and SEN450 inhibits the SMO function while HPI1 inhibit the GLI. (B) Notch Pathway – The binding of Delta like ligand (DLL) or Jagged ligand activates ADAM and several other proteins that cleaves the Notch intracellular domain (NICD) from the notch receptor. The NICD then moves to the nucleus and helps in the expression of Notch related genes. The therapeutics Rova-T and DLL4-Fc inhibits the DLL ligand while Tarextumab inhibits the notch receptor. (C) FGF pathway – The Fibroblast growth factor receptor (FGFR) is activated when FGF is bound to it. FGFR then phosphorylates Fibroblast Growth Factor Receptor Substrate 2 (FRS2) at multiple sites. This initiates several signalling cascades where Ras and Phosphoinositide 3-kinases (PI3K) being the prominent ones. Their end effectors ERK and mTOR help in transcription of target genes. The therapeutics against this pathway PD173074 and Erdafitinib target the FGFRs.

4.1.1 Role of Hedgehog pathway in SCLC

During the development of the lung, Hh signalling pathway is crucial for the regulation of branching morphogenesis in a paracrine manner. However, in normal adult lungs, this signalling pathway gets reactivated during severe tissue damage [22] and becomes necessary for the stem cell compartments [26]. In the case of SCLC, it is found that some of the cells express Shh while the adjacent cells express PTCH1. This indicates that similar to lung development and repair, SCLC cells also have an active Hh signalling in a paracrine manner [27]. It was supported by the fact that there was a significant reduction in the SCLC growth upon treatment with 5E1 Shh-N monoclonal antibody [22] indicating the importance of this pathway in tumor growth. However, there is no evidence of the change in copy number of the hedgehog genes till date [28]. In a study by Park and his collaborators, it was found that Hh signalling plays an important role in SCLC right from its initiation to its maintenance indicating that both initial and advanced stages of SCLC tumors are dependent on an active Hh signalling [13]. In this study, it was observed that upon deletion of SMO in Trp53 and Rb1 negative lung mutant cells, the initiation of SCLC was suppressed. Also, they could successfully inhibit the tumor growth post-chemotherapy, in both human and mouse models by inhibiting the Hh pathway.

4.1.2 Therapeutics targeting Hedgehog pathway in SCLC

As already discussed, the Hh pathway plays a crucial role in SCLC right from its initiation to its development, it has been used as a potential target. The majority of the studies are directed against the SMO protein that plays a vital role in activating the Hh pathway. Cyclopamine is one such compound that selectively binds to SMO to inhibit its function by causing its conformational change which is very similar to that caused by PTCH1 activity [29] (Table 1). Vismodegib, a novel small molecule inhibitory drug that is directed against SMO is widely used to inhibit the Hh pathway after noticing its promising results in the case of advanced Basal cell carcinoma [30] (Table 1, Ref. [29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44]). However, there was no significant improvement observed in the ED-SCLC patients when Vismodegib was administered along with standard chemotherapy in ED-SCLC [31]. Apart from this, the SMO inhibitors namely Erismodegib and Vismodegib did not show any significant results on 23 different SCLC cell lines when given along with chemotherapy. Similar findings were observed even with the treatments of HPI1 and SEN450, specific inhibitors for GLI and SMO respectively (Table 1). The compounds erismodegib, HPI1, and SEN450 showed a better result in the case of MYC amplified cell lines [32] indicating a critical role of MYC in this mode of treatment (Fig. 1A).

Table 1.Therapeutics that have been tested against SCLC that target developmental signalling pathways.

S. No	Name	Target pathway	Mechanism of Action	Current Status in SCLC	References
1	Cyclopamine	Hedgehog	It inhibits SMO by causing a confirmational change that is similar to the PTCH1 activity when the Hh ligand is present.	Not enrolled in clinical trials	[29]
2	Vismodegib	Hedgehog	Acts as SMO antagonist and inhibits the downstream signalling of the Hh pathway.	Phase 2 completed	[31, 39]
3	Erismodegib	Hedgehog	SMO antagonist that can induce apoptosis in cancer cells.	Phase 1 completed	[40, 41]
5	HPI1	Hedgehog	Induces structural changes on GLI that inhibit the gene expression in SUFU deactivated or GLI overexpressing cells.	Not enrolled in clinical trials	[32, 42]
6	SEN450	Hedgehog	SMO antagonist.	Not enrolled in clinical trials	[32, 43]
7	DLL4-Fc	NOTCH	Reduces Notch1 signalling in DLL4 expressing cells. This halts the early metastasis of cancer cells.	Not enrolled in clinical trials	[33]
8	Tarextumab (OMP-59R5)	NOTCH	Targets and inhibits the Notch2/3 signalling. It is a human IgG antibody specific for Notch receptors.	Terminated in Phase 1b	[34]
9	Rova-T (Rovalpituzumab Tesirine)	NOTCH	It is an antibody-conjugated drug that targets DLL3. Once bound, the linker is cleaved releasing the drug into the cytoplasm. The drug enters the nucleus and binds to the DNA leading to cell cycle arrest.	Phase 3 completed	[35, 44]
10	Erdafitinib	FGF	Binds and inhibits the function of FGFRs. Mainly inhibits the phosphorylation which ultimately inhibits the downstream signaling.	Not enrolled in clinical trials	[37]
11	PD173074	FGF	Binds and inhibits the function of FGFRs.	Not enrolled in clinical trials	[36]
12	XAV939	WNT	Downregulates $\beta$-catenin which reduces the growth of cyclin D1.	Not enrolled in clinical trails	[38]

4.4 Other developmental signalling pathways in SCLC

As already mentioned, there is no clear understanding of how SCLC progresses in the human body. Thus, there is still a lot more that needs to be identified as to which developmental pathways are active during the development of this cancer. Most of the research in this area is concentrated on the above-mentioned pathways. However, there is some evidence for the activation of other developmental pathways as well. For example, RNA-sequencing data revealed that the WNT signalling pathway was mutated predominantly in the case of SCLC patients with recurrent disease [79]. However, this study was performed on a very small population of 12 patients who have previously undergone treatment with platinum chemotherapy. Thus, it can’t be clearly said if these mutations are a part of disease progression or they are generated due to the treatment [80]. Prior to this study, the evidence for activation of WNT signalling in SCLC was indirectly given by the overexpression of the SOX-2 gene that in turn regulates the expression of certain oncogenes like n-MYC, WNT-1, WNT-2, and NOTCH1 in human lung adenocarcinomas [81]. Further, there was a clear indication that downregulation of SOX-2 decreased tumorigenesis and induced apoptosis [81]. Thus, it can be inferred that SOX-2 is responsible for the activation of pathways like WNT and NOTCH which in turn induce tumorigenesis. A study by Pan and his group have demonstrated that XAV939, a small molecule inhibitor of the WNT signalling pathway could significantly inhibit the proliferation of the SCLC in vitro [38] (Fig. 2A).

Fig. 2.

Role of Hippo, TGF-$\beta$ and WNT pathways in SCLC and their therapeutic interventions. (A) WNT pathway – The WNT ligand when bound to the frizzled receptor and LRP5/6 translocates Axin and GSK- $\beta$ towards the plasma membrane. This results in accumulation of $\beta$-Catenin in the cytoplasm and then its transport to the nucleus where it regulates the gene expression. The transport of $\beta$-Catenin to the nucleus is prevented by XAV939, a small molecule inhibitor of WNT pathway. (B) TGF-$\beta$ Pathway – The ligand TGF-$\beta$ binds to the receptor TGFR that activates it. The activated receptor phosphorylates receptor mediated SMADs (R-SMAD) that binds with SMAD4 to function as a transcription factor. (C) Hippo pathway – The ligand binding at the Receptor Tyrosine Kinases (RTK) and G protein-coupled receptors (GPCR) releases a signal that inhibits phosphorylation of proteins like MST1/2, SAV1, LATS1/2, MOB1. This releases the LATS1/2 and MOB1 complex inhibiting yes-associated protein 1 (YAP) that is a transcription factor can regulate important cellular functions like apoptosis.

The transforming growth factor-$\beta$ (TGF-$\beta$) signalling pathway is reported to be upregulated in various pulmonary diseases [82]. This pathway inhibits early-stage tumor progression but in later stages, it promotes tumor growth. In the case of SCLC, the inhibition of this pathway reduced the migration and invasion of the cancer cells [83]. This was confirmed by the downregulation of miR-886-3p miRNA which is in turn responsible for the post-transcriptional suppression of TGF-$\beta$1 and PLK1 [84] (Fig. 2B).

The overexpression of WW domain-binding protein 5 (WBP5) further indicates the involvement of another developmental pathway like Hippo in SCLC [85]. Activation of this pathway resulted in multidrug resistance, enhanced tumor growth, and shorter survival in SCLC patients. Later on, it was revealed that this resistance was mediated by the Hippo pathway through the inhibition of phosphorylation of key proteins like MST2 (mammalian Ste20-like kinase 1) and YAP1 (Yes-associated protein -1) [86]. This supports the idea that the Hippo pathway plays a role in developing multidrug resistance in SCLC. Recent research also showed that the Hippo pathway plays a role in radiation resistance in SCLC patients. It was found that MST2 had no significant role but YAP1 along with CD133 had a profound role in developing radiation resistance in SCLC [87] (Fig. 2C).

These findings clearly show that developmental signalling pathways are involved in key aspects of tumorigenesis in SCLC progression and also contribute to its neuroendocrine behavior. However, there are lots of grey areas that need to be addressed properly in order to consider the components of these pathways as drug targets.

In recent times, cancer treatment using immunotherapy is gaining importance [88]. The use of immunotherapy in SCLC is employed alongside etoposide or other chemotherapeutic agents that are used for SCLC treatment. The immunotherapy treatment through PD-L1 inhibition is the current method for SCLC treatment. Studies indicated the cross-talk of this signalling with developmental signalling pathways like hedgehog and WNT in cancers like breast cancer and gastric cancer. In triple-negative breast, it was observed that WNT inhibitors play a role in the down-regulation of PD-L1, while the Hedgehog pathway favours the expression of PD-L1 in gastric cancer [89, 90]. However, the exact mechanism by which these pathways are interrelated is not yet known. In addition, immunotherapy-mediated regulation of signalling pathways in the case of SCLC is also need to be deciphered.

SCLC is a lethal malignancy with a rapid doubling time. There is no complete understanding of how this cancer initiates and progresses in the body. The patients respond well to initial therapy, but majority of them show relapse within a few months’ time. Etoposide and topotecan along with platinum-based chemotherapy are being used as the first-line and second-line drugs respectively since 1970’s. There has been no significant improvement in the treatment strategies over few decades. However, recent improvement in the radiation technology has modestly increased the survival rate for both limited and extensive stage SCLC. Although molecular anomalies associated with SCLC are known to the oncologists, efforts to target these aberrations over a decade have been consistently unsuccessful resulting in dismal prognosis for SCLC. As a result, SCLC has been enlisted along with other difficult-to-treat cancers in the Recalcitrant Cancer Research Act of 2012 instructing National Cancer Institute (NCI) to allocate funds to deal with it. There is always a new hope that the improved understanding of the underlying molecular mechanisms may result in development of new therapeutics for SCLC. Thus, there is a need to identify more effective targets and also gain knowledge about the progression of this cancer. In this respect, research on the developmental signalling pathways has gained importance. There have been some interesting outcomes that demonstrate the importance of these pathways in initiation, growth, metastasis, invasion and progression of SCLC. The NOTCH and Hedgehog pathways were the most explored developmental pathways in SCLC. In other types of cancers, it has been observed that developmental pathways like FGF, Hippo, TGF-$\alpha$ and WNT had a profound role in tumorigenesis. But these pathways have not been studied extensively in SCLC. Preliminary studies gave some positive results in controlling the tumor growth. However, these compounds have not yet been tested in humans or if tested, the sample size of patients was very small to draw any conclusions from the study. Hence, we believe that an in-depth study in these developmental signalling pathways can throw more light into the progression and initiation of SCLC. Also, the crosstalk among these pathways can help us gain a better understanding about the neuroendocrinal switch and metastatic features of SCLC. Therefore, it is very much necessary to decipher the signalling cascades of the developmental pathways in more detail to find potent drug targets for successful treatment of SCLC. In view of the above, the current article provides the first comprehensive review about the available information on the role of developmental pathways in SCLC which can ultimately help in better prognosis and treatment of this cancer.

Conceptualization—SKS, PMS, PKV; Formal analysis and investigation—PMS, SKS, PP; Writing - original draft preparation—PMS, AS, PP, NC; Writing - review and editing—SKS; Resources—PMS, AS, PP, SKS; Supervision—SKS, PKV, AKS, BP.

Not applicable.

Pingali M. Shivapriya and Anirudh Singh are thankful to MHRD, Govt. of India for fellowship. We are thankful to IIIT Allahabad for providing research facility. We are thankful to Arka Banerjee at department of Microbiology and Immunology, Weill Cornell Medicine and Ayush Amod, Dept. of Applied Sciences, IIITA for critical reading of the manuscript.

This research received no external funding.

The authors declare no conflict of interest.