Heat shock proteins in tumor resistance
Other sensitizers that have been studied in potential combinational therapies are the heat shock proteins (HSPs). HSPs are chaperones with cytoprotective role into the cells responsible by proper folding of proteins. HSPs are classified according to their molecular weights in Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small HSPs [
96]. Among these proteins, Hsp90, 70, 40 and 27 have received special attention in studies that aim to inhibit tumor growth and progression. Hsp70 and Hsp90 are proteins directly involved in refolding proteins; Hsp40 transfers the unfolded protein to Hsp70 by complexing with HIP (Hsp70 interacting protein) and stimulates the ATPase activity of Hsp70; Hsp27 prevents aggregation of unfolded proteins into the cytoplasm [
97,
98].
Jeong
et al.[
99] demonstrated the association between Hsp90 inhibition and decrease in proliferation of a non-small cell lung cancer (NSCLC) cell line resistant to gefitinib. Other study using NSCLC cells showed that treatment with Hsp90 inhibitor ganetespib induced loss of EML4-ALK gene rearrangement found in this type of tumor and depletion of multiple oncogenic proteins [
100]. Hsp90 inhibitor CH5164840 showed antitumor activity on NSCLC cell lines and enhanced the efficacy of erlotinib. The combination of these compounds suppressed ERK signaling in a cell line resistant to erlotinib [
101]. Hsp90 inhibition lead to apoptosis induction by mitochondrial pathway in melanoma, cervix, colon, liver and lung cancer cells and induced apoptosis in cells overexpressing Bcl-2 [
102].
Hsp70 is currently upregulated in several cancer types and can be induced by drugs that trigger the heatshock pathway signaling. Hsp70 protects normal and tumor cells from death by binding to Bax and Apaf-1 after a stress stimulus [
103,
104]. A barrier to the complete successful of Hsp90 inhibition in treatment of cancers is that its inhibition increases the Hsp70 expression [
105]. Some authors showed that inhibition of Hsp70 alone is few less or ineffective to cause cell death in tumors, nevertheless it could enhance the antitumor effects of other drugs a great coadjuvant in the treatment of cancers [
106‐
108]. The Hsp40 group has a role as co-chaperone for Hsp70 and indirect regulator of Hsp90 and it contains the greatest number of members. In fact, the diversity of structures and functions of the group makes targeting Hsp40 very challenging (for review, see Sterrenberg
et al.[
109]).
Different works showed the relationship between Hsp27 and direct activation of Akt, increasing the cell survival signaling pathway by regulating negatively pro-apoptotic proteins in different models [
110,
111]. Kim
et al. [
112] demonstrated that inactivation of ERK/p90RSK/HSP27 cascade in SK-OV-3 cells by melatonin enhances cisplatin-induced apoptosis. Hsp27 inhibition by quercetin also reduced the viability of A549 cells when used in combination with cisplatin or gemcitabine when compared to these drugs alone showing the role of Hsp27 in chemoresistance [
113]. Other studies showed the antitumor effects of Hsp27 inhibition in combination with other drugs (for review, see McConnell & McAlpine [
114].
Monolayer x 3D cell culture
Cell culture in a monolayer system, also known as two-dimensional culture (2D), does not maintain the same features found
in vivo. The development of other culture systems are growing up to achieve one that better mimics the
in vivo cellular features, very important to improve studies about cancer disease, for example, in the evaluation of drug effects in cancer cells [
115]. The three-dimensional culture (3D) is a type of culture that increases cell interactions with other cells and with the ECM, which is closer to
in vivo conditions [
116,
117].
The increased cell-cell or cell-matrix interactions observed in 3D culture can: a) augment cell differentiation [
118‐
120]; b) change cell signaling in response to ECM compounds [
121]; c) modify the gene expression pattern [
122,
123]; and d) alter the expression of proteins linked to cell adhesion to matrix (integrins) and cell-cell adhesion (cadherins) [
124]. The expression of integrin and E-cadherin distribution in spheroids were similar to
in vivo results [
125,
126].
There are several models of cell culture in a 3D environment, such as: multicellular spheroids [
127,
128], microcarrier beads, synthetic (synthetic gels) or natural materials (matrigel, a gel with ECM obtained from mouse sarcoma cells in culture, and type I collagen) that provide cell growth in a three-dimensional system and organotypic explant culture [
121].
The 3D culture may be a good model for both basic and applied research. Cancer cells culture in a 3D system is very interesting to study cancer disease, for example, evaluating the effects of drugs in these cells. Cells maintained in a 3D environment are organized in multiple layers that confer a biological barrier to drug diffusion, like small avascular tumor aggregates observed
in vivo[
121]. Fourré
et al.[
129] cultivated fibrosarcoma cells HT-1080 in a 3D culture type with type I collagen and showed that doxorubicin cell penetration took about 1 more hour compared to cells grown in a 2D system. Other works showed similar results with the same drug: Yip & Cho [
130] found that cells cultured in the presence of collagen hydrogel had higher cell viability and Millerot-Serrurot
et al.[
131] observed that ECM protected cancer cells from anti-migratory effect of doxorubicin. However, in these cases, the decreased drug penetration was due to mechanical resistance and not cell chemoresistance [
132].
Some cell types become more resistant to cell death via apoptosis while they are in contact with other cells or with the ECM, as it occurs in 3D cell cultures. For example, cell adhesion mediated by integrins leads to increased expression of integrin receptors and fibronectin, such as VLA-4, which seems to be related to apoptosis resistance [
133,
134]. Also, in some 3D models in which cells became polarized after the contact with an ECM similar to the basement membrane, the expression of beta4 integrin was associated with resistance to apoptosis [
135]. This type of resistance, also known as CAM-DR (cell adhesion mediated drug resistance), is seen today as a target to anti-cancer therapies [
136,
137].
Studies have shown that cells are more resistant to drugs when grown in 3D cultures than when they are in monolayer. Longati and co-workers [
138] tested the resistance of pancreatic ductal adenocarcinoma cell line (PDAC) in 2D and 3D cultures. The cells are more resistant to gentamicin, CD5, CB7, CB13, Act16412 and GANT61 when they are in 3D cultures. Human ovary cancer cells (SKOV3 cells), when cultured in 3D to mimic ascites, form cell aggregates resistant to paclitaxel [
139,
140]. This resistance could be due to high expression levels of KLK4 (high tumor kallikrein-related peptidase 4) [
140]. Similarly, stem cells isolated from SKVO3 cultures, when cultivated in a 3D environment with basement membrane extract scaffold, are resistant to docetaxol, cisplatin, carboplatin and 5FU. In cells grown in this model of 3D culture, the expression of ABCB1 and ABCG2 are increased and could be related to resistance to the drugs tested [
141]. Lung cancer cells also become resistant to bortezomib when in 3D cultures [
142], as well as MCF-7 cells which become resistant to 5-FU in specific stages of spheroid formation [
143].
Some genes related to drug resistance in 2D cultures are over expressed in 3D cultures and could be involved with drug resistance in these models. Among these genes, we can mention BCL-2 family members, ABCG2 and ABCB1, CP78 and KLK4. Other interesting hypothesis for drug resistance in 3D cultures is related to the increased cell adhesion and matrix elements synthesis in these models, making it difficult for drugs to penetrate in the spheroids and reach all the cells [
138]. However, some studies show that certain drugs such as doxorubicin can penetrate in spheroids and be incorporated in cells nuclei within these large structures. In this case the drug retention does not depend on MDR1 bombs, but the resistance seems to be related to the expression of Bcl-2 family members [
31].
Fourré and co-workers [
129] also show in collagen rich models that doxorubicin and anthracyclins take more time to be detected in cell nuclei and that in these cases it takes longer treatments to reach similar cellular responses to those seen in 2D cultures.
Nirmalanandhan and co-workers [
144] tested 10 different drugs in lung cancer (A549) and bronchioalveolar cancer (H358) cells cultured in 2D and 3D models with type I collagen. When tested in H358 cells, 8 of the 10 drugs needed different concentrations to reach the same effects in 2D and 3D. In A549 cells 7 of the 10 drugs showed similar effects but in different concentration. The results depend on the cell line and on the drug class, and show that more studies should be done to determine if the mechanisms of chemoresistance in 3D models share the same features of the mechanisms observed in 2D cultures. Moreover, it is important to evaluate if the cell responses to drugs in 3D are more similar to what happens
in vivo than those observed in 2D cultures, making it a new way to test drugs and to evaluate chemoresistance. A summary of studies with drug resistance in 2D and 3D cell cultures is presented in Table
1.
Table 1
Effects of some drugs and cancer cell mechanisms of drug resistance in monolayer and in three-dimensional cell cultures
Doxorubicin/Paclitaxel/Tamoxifen/ | MCF-7 (breast carcinoma) | Antiproliferative effect increased in 2D; cells more resistant to drugs in 3D. | Increased ECM production in 3D models, difficulting drug diffusion. | |
AG1478 | SW-480, HT-29, DLD-1, LOVO, CACO-2. COLO-205, COLO-206F (Colorectal Cancer Cell Lines) | Cell viability decreased in a dose-dependent way in 2D and 3D, but more clearly in 2D. | Cells in 3D showed decreased expression of EGFR. ECM signaling can be involved too. | |
5FU/ Docetaxel/ Cisplatin/Carboplatin | SKOV-3 cell line (epithelial ovarian cancer). Stem cells were selected from these cultures. | Antiproliferative effect increased in 2D. Cells in 3D models more resistant to drugs. | Decreased apoptotic induction in 3D. Increased expression of ABCG2 e ABCB1 in 3D. | |
Paclitaxel | SKOV-3 (epithelial ovarian cancer) | Increased expression of KLK4 favors multicellular aggregates formation and these are more resistant to drug. | Increased expression of KLK4. | |
Doxorubicin/ Cisplatin | SW1353, CH2879, JJ012, OUMS27 (chondrosarcoma cell lines) | In 3D models doxorubicin can reach cell’s nuclei but cells are more resistant to the drug. | Drug penetration is not dependent on MDR activity. Bcl-2 members are important to resistance. | Van Oosterwijk et al.[ 29] |
Taxol/Cisplatin | HEY, A2780, SKOV3, OVAC429 (human ovarian cancer) | Cells cultured in 3D are more resistant to taxol treatment. | Cells from some cell lines did not arrested in G2-M after taxol treatment. | |
Doxorubicin | HepG2 (human hepatocellular liver carcinoma) and 3T3-J2 (fibroblasts murine stromal cells) | Increased cell viability in 3D. 3D heterospheroids are more resistant than 2D and homospheroid models. | Stromal fibroblasts and collagen hydrogel culture system provides more resistance. | |
Doxorubicin | HT1080 (human fibrosarcoma) | In 2D doxorubicin decreased cell migration, and in 3D the drug did not affeted cell migration. | EMC proteins in a 3D configuration are able to protect cancer cells from the antimigratory effect of doxorrubicin. Environment-mediated drug resistance. | |
Docetaxel/Cisplatin/5-FU/Gemcitabine/ Camptothecin | H460, A549, H1650 (lung cancer) | All drugs showed increased IC-50 in 3D. | Caspase-3 is decreased in 3D. Drugs could not penetrated into cells in 3D and apoptosis was decreased. | |
Cancer stem cells
The concept of cancer stem cell (CSC) was stated based on the organization of multicellular organisms presenting somatic stem cell populations that give rise to committed progenitors which are able to differentiate into mature cells. Normal cellular hierarchy comprises stem cells that progressively generate more restricted progenitor cells, yielding all the mature cell types that constitute a particular tissue. Cancer would simulate organ development, exhibiting a similar hierarchy with different cell populations, including the CSCs, associated to high drug resistance.
In the strict sense, CSCs and tumor initiating cells (TICs), i.e., cells that acquired the tumor promoting mutations are conceptually different. CSCs (and not other tumor cells) would be the only cells capable of sustaining tumorigenesis due to their self-renewal and asymmetric division abilities. TICs are defined as cells capable of initiating a tumor in immunocompromised mice [
145]. However, the terms CSCs and TICs have being indistinctly used to refer to the small cellular subpopulation (0.01-1% of total tumor cells) first described in leukemia and then in breast cancer and others solid tumors [
16,
17,
146,
147]. These cells are able to induce cancer when transplanted to immunodeficient mice, have drug resistance and self-renewal ability. It is believed that the resistance of CSCs to currently used chemotherapeutics is a major contributing factor in cancer recurrence and later metastasis development.
According to their phenotypes, CSCs can be identified and isolated by means of 4 main methodologies: a) cell sorting by flow cytometry using specific cell surface markers [
148,
149]; b) assessment of aldehyde dehydrogenase (ALDH) activity [
150]; c) cell sorting of side-population (SP) phenotype by Hoechst 33342 exclusion [
151]; d) spheres isolation, since CSCs are able to form floating colonies from a single cell more efficiently than their progeny [
152] and to grow as spheres in non-adherent culture conditions [
153].
The most commonly used surface markers are CD44+ and CD133+ [
17,
146]. CSC phenotype in leukemia was associated with CD44+/CD38- cells [
16]. CD44 is a transmembrane glycoprotein believed to be activated in a wide range of tumors in which it plays a critical role in cancer cell adhesion, migration, invasion and survival [
154]. It is a multifunctional cell surface adhesion molecule associated with cell-cell and cell-matrix interaction. CD44+ has identified cells with the ability of give rise to new tumors
in vivo, in different types of cancer. Patient samples of head and neck squamous cell carcinoma (HNSCC), for example, contain a heterogeneous population of cancer cells and the small subpopulation CD44+ contained most of the CSCs, evidenced by its tumorigenic potential in immunodeficient mice [
155].
CD133 (prominin-1 or AC133) was originally described in human hematopoietic stem cells and has subsequently been used as a marker to isolate CSCs from many tumor types. It is a member of the pentaspan transmembrane glycoprotein family involved in a variety of cellular activities. CD133 is found to be selectively localized in microvilli and other plasma membrane protrusions irrespectively of cell type and interact with membrane cholesterol. Wnt, Notch, TGFβ1, Line-1 and methylation regulate its expression. CD133 is involved in energy metabolism and in autophagy, which are beneficial for the survival of cancer stem cells.
ALDH activity is an important functional marker of normal and malignant stem/progenitor cells. ALDHs contribute to drug resistance through detoxification of many cytotoxic agents provided that aldehydes are generated by several metabolic processes (reviewed by Marchitti
et al.[
156]). Increased ALDH activity in hematopoietic stem cells, for example, contributes to metabolize and detoxify cyclophosphamide [
157]. The ALDH family of enzymes comprises 19 isoforms that can be found in different cell compartments: nucleus, cytoplasm or mitochondria. In a retrospective analysis of breast cancer patient samples, ALDH1A1, but not ALDH3A1, expression was found to be predictive of tumor responsiveness to cyclophosphamide and other oxazaphosphorines treatment [
158]. In support of this potential role for ALDH in CSC resistance to chemotherapeutics, CSC enrichment was observed in colorectal cancer xenograft tumors after cyclophosphamide treatment, and this was correlated with enhanced ALDH1A1 expression and enzymatic activity.
Antibodies against the ALDH enzyme family are available, but the vast majority of studies have used cell-sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the Aldefluor assay and sorted by fluorescence-activated cell sorting. This approach was used by Cheung
et al.[
159] in one of the first studies isolating ALDH+ cells from acute myeloid leukemia. ADLH+ enriched cell population was similarly isolated from breast cancer [
160]. In both studies, the isolated cells presented self-renewal ability and high tumorigenic potential. ADLH+ cells with CSC phenotype were isolated from several hematopoietic and solid tumors including lung, liver, bone, colon, pancreatic, ovarian, head and neck, and prostate cancers.
The aldefluor activity specific for the CSCs of these cancers has been attributed to ALDH1A1 and so prognostic studies have been targeted to this isoform. However, Marcato
et al.[
161] claim that ALDH1A3 and other ALDH isoforms activities contribute to aldefluor positivity.
Additionally, ALDHs participate in ester hydrolysis and act as antioxidant. Enzymatic aldehyde dehydrogenase activity of some specific isoforms is important for the preservation of undifferentiated stem cells, by interfering with the biosynthesis of endogenous retinoic acid (RA) through the oxidation of all-trans-retinal and 9-cis-retinal. The cytosolic isoform ALDH1A1, associated with metabolism and detoxification of cyclophosphamide, plays a role in the differentiation of several cell types through the oxidation of retinal to RA [
156].
RA modulates biological processes like cell proliferation, differentiation, cell cycle arrest and apoptosis. All-trans-RA is used to treat acute promyelocytic leukemia, since it induces differentiation of immature leukemia blasts into terminally differentiated granulocytes, leading to a clinical remission in approximately 90% of patients. Based on these results, retinoic acid effects are being studied in other cancers and cancer cell lines. The combined use of RA (0.1 μM) and cAMP (1 mM), an important second messenger, improves the responsiveness of hepatocarcinoma cell line (HTC) to RA treatment. RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, treatment with RA and cAMP increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased, showing that the combined use of RA and cAMP is more effective in inducing differentiation [
162].
The use of the vital dye Hoechst 33342 exclusion as a method to isolate normal hematopoietic stem cells was proposed by Goodell
et al.[
163]. The method defines an easily identifiable and highly reproducible small cell population (0.1% of bone marrow cells), presenting stem cell phenotype. The Hoechst-exclusion SP assay has the advantage of measuring a functional parameter of the cells. Widely used in hematological malignancies, the methodological approach requires additional steps such as enzymatic cell disaggregation for solid tumor samples analyses [
164]. Both normal and cancer stem cells express the ABC transporters. The ABC domain of these transmembrane proteins allows ATP binding and hydrolysis, and the ABC protein can function as receptor, channel and multidrug transporter, participating in the efflux of small molecules. These pumps detoxify cells through the efflux of cytotoxic agents, being responsible for the exclusion of the dye Hoechst 33342.
SP cells were isolated and characterized in most human cancers including HNSCC, bladder, ovarian, pancreas, lung, hepatocellular carcinomas, osteosarcoma and Ewing’s (for review, see Tirino
et al.[
164]). SP cells were consistently shown to represent a stem cell-enriched population. Compared to non-SP cells, a smaller number of SP cells are able to grow as tumors when injected in immunodeficient (NOD/SCID) mice.
Breast cancer cell lines, like MDA-MB-231 and MCF-7, show anoikis-resistance in drug treatments with doxorubicin. The SP cells fraction in the anoikis-resistant cancer cells seems to be higher than the parental cells [
165]. There are reported mechanisms that contribute to SP chemoresistance including relative quiescence, expression of ABC transporters and/or MDR1, a more efficient DNA-repair capability, and the elevated expression of anti-apoptotic proteins.
The high tumorigenic efficiency of SP cells is associated with drug resistance and with the presence of other CSC markers, such as ALDH+, CD133+ or CD44+. He et al. (2013) proposed the phenotypical modulation of CSCs, which involves the conversion of SP to non-SP cells (and vice versa), to be under PI3k/AKT and β-catenin /CBP signaling pathway. Beta-catenin accumulation enhanced the transition from non-SP to SP phenotype, and siRNA against any of the downstream signals abrogated the conversion of non-SP to SP cells in breast and bladder cancer cell lines.
Other method for CSC isolation was based on the observations of Reynolds
et al.[
166] that some cells of the central nervous system were able to grow in suspension when plated on non-adherent surface, forming structures named spheres or neurospheres. These floating colonies were able to self-renewal, once when enzymatically dissociated, they originated several new spheres. Their stem cell phenotype was confirmed by the ability to originate different cell types under adequate stimulation (astrocytes, neuron or oligodendrocytes). The floating sphere formation is consequence of the ability to grow independently of surface anchorage and resistance to anoikis associated with high clonogenicity, features shared by both normal and cancer stem cells.
Spheres were grown from different human cancer samples and cancer cell cultures and they consisted mainly of CSCs (review in Alamgeer
et al.[
167]). SCLC and NSCLCCD133+ cells, when submitted to long-term culture as spheres, could modify their phenotype to CD133- cells [
153]. The phenotype modulation of CSCs is important to define more efficient therapies. MCF-7 cell line long-term spheroids showed high degree of cell differentiation, organizing duct-like structures [
119].
The CSC model represents a very important tool in cancer biology, especially in relation to the problem of drug resistance. CSC/TIC cells may exist independently of the described markers and the cellular plasticity may be much more relevant. Nevertheless, the current identification of markers and pathways is already underpinning some novel developments in therapeutic strategies for patients with cancer.